Evaluation of chitosan oligosaccharide as an alternative treatment to mitigate Fusarium graminearum and mycotoxin contamination in wheat

Evaluation of chitosan oligosaccharide as an alternative treatment to mitigate Fusarium graminearum and mycotoxin contamination in wheat | 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 Research Article Evaluation of chitosan oligosaccharide as an alternative treatment to mitigate Fusarium graminearum and mycotoxin contamination in wheat Gabriel Ferreira Paiva, Lara Lorrayne Silvestre de Andrade, José Maria da Silva, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7915619/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Fusarium head blight (FHB), caused by members of the Fusarium graminearum species complex, is one of the most serious wheat diseases worldwide, capable of causing severe yield losses and affecting grain quality. Methods available for FHB management include cultural practices, sowing of resistant cultivars, chemical control, and biological control. In the present study, we evaluated the effect of chitosan oligosaccharide on the control of fungal growth, seed infection, disease symptoms and mycotoxin contamination under both controlled environments and in the field. In the in vitro conditions, chitosan oligosaccharide directly affected fungal growth at different levels depending on the concentration used. When applied as a seed treatment, both tebuconazole and chitosan oligosaccharide reduced the incidence of F. graminearum and preserved seed germination potential of infected seeds. In the field, there was no significant difference in disease severity and DON in grains between the unsprayed check and the fungicide, regardless of the doses or times of chitosan oligosaccharide application. The results of this study suggest that chitosan oligosaccharide can potentially be used as a tool in the integrated management of the disease, establishing a basis for future studies to explore its use associated with chemical products, aiming to increase the efficacy of FHB control in wheat. Wheat scab Triticum aestivum Alternative control Integrated management of plant diseases Figures Figure 1 Figure 2 Figure 3 Introduction Fusarium head blight (FHB), caused by members of the Fusarium graminearum species complex, among which Fusarium graminearum s.s. is one of the most dominant species, is one of the important diseases of wheat worldwide. Losses due to FHB in wheat and barley crops in the United States is estimated at around $ 2.7 billion. The disease has been considered a resurgent problem worldwide since the early 1990s, posing significant challenges to agricultural production and food security, and grain quality due to the accumulation of mycotoxins, such as the B-trichothecene deoxynivalenol (DON), nivalenol (NIV) and their acetyl-derivatives (Duffeck et al. 2020 ; Huang et al. 2020 ; Figueroa et al. 2018 ; McMullen et al. 2012 ; Del Ponte et al. 2009 ; Nganje et al. 2004 ; Tralamazza et al. 2016 ). FHB is considered a floral and monocyclic disease, where primary infection occurs from inoculum surviving in plant debris. The main symptoms are bleaching of spikelets with the presence of pinkish or orange-ish signs of the pathogen in the base of the glumes, leading to kernel infection, yield reduction, and contamination of tissues and grains with mycotoxins (Snijders 2004 ; Gilbert and Haber 2013 ). The resurgence of FHB s, in southern Brazil, a subtropical environment, has been associated mainly with climate variability, while the role of within-field local inoculum has been questioned, which contrasts with studies in the temperate regions climatic factors (Markell and Francl 2003 ; Del Ponte et al. 2005 , 2009 ; Duffeck et al. 2020 ). The management of FHB aims to reduce disease intensity as well as mycotoxin levels to meet the maximum tolerated limits in wheat products as established elsewhere for each country, including Brazil (ANVISA 2011, 2017, 2022). There are several methods available for the management of FHB, including cultural practices, resistant cultivars, chemical control, biological control, and others, with the the integration of all these practices being more more effective than any one applied in isolated (Wegulo et al. 2015 ; Chen et al. 2019 ). Chemical control is one of the most used measures for an effective FHB control. Demethylation inhibitor fungicides (DMI) are the most widely used for the control of disease, and the reduction in the accumulation of mycotoxins (Paul et al. 2008 ; McMullen et al. 2012 ; Wegulo et al. 2015 ). Overall, the fungicides used to control FHB show variable efficacy. In a meta-analytic study in North America, it has been shown a large variation of control efficacy across the trials, with performances ranging from 32 to 50% for different the DMI fungicides tebuconazole, propiconazole, metconazole, and prothioconazole (Paul et al. 2008 ) and in Brazil for triazole and benzimidazole (Machado et al. 2017 ). With the recurrent use of fungicides in the management of this disease, there is a risk to select resistant individuals within the F. graminearum population. In fact, in the state of New York and in Henan Province in China, tebuconazole less sensitivity isolates of F. graminearum have been reported (Spolti et al. 2014 ; Chen et al. 2021 ). Therefore, new control methods must be developed. The use of other products such as chitosan derivatives may be a viable option to integrate management targeting disease and mycotoxin control. Chitosan is a polymer of D-glucosamine derived from the deacetylation of chitin which is found naturally in the cell wall of fungi and also in the exoskeleton of crustaceans, being considered the second most abundant polysaccharide in nature (Berger et al. 2011 ; Almeida et al. 2019 ). Chitosan has been studied for its ability to increase plant tolerance to stress and to activate plant defense responses (Katiyar et al. 2015 ; Suarez-Fernandez et al. 2020 ; Saberi Riseh et al. 2022 ). Previous studies have already reported the effect of chitosan inhibiting F. graminearum mycelial growth and conidia germination in vitro (Kheiri et al. 2016 ; Xu et al. 2007 ; Deshaies et al. 2022 ). Chitosan oligosaccharides are the degraded products of chitin or chitosan by acid hydrolysis and/or enzymatic degradation, and it is considered a chitosan oligomer, and compared to chitosan, has a higher water solubility and lower viscosity (Yin et al. 2016 ; Muanprasat and Chatsudthipong 2017 ; Naveed et al. 2019 ). The chitosan oligosaccharide, after applied to the plant surface, is recognized by a receptor on the plant cell membrane, followed by signal transfer and amplification, activation of responsive genes, accumulation of responsive proteins, induction of defense-related secondary metabolites, and finally the plant response, such as hypersensitivity responses, production of phytoalexins and reinforcement of cell walls (Yin et al. 2010 ). This study aims to investigate the potential of chitosan oligosaccharide, a chitosan derivative, as an alternative control product, sprayed alone, for the control of FHB intensity as well as DON accumulation in wheat grains. Materials and Methods Fungal isolate and inoculum preparation A F. graminearum DON/15ADON-producing isolate (CML 3066), representative of the dominant population in Southern Brazil, was chosen for this study (Del Ponte et al. 2015 ; Wood et al. 2020 ). The isolate was grown on potato dextrose agar (PDA) at 25°C with a 12-h dark/light cycle for seven days. Afterwards, mycelial plugs were transferred to plates containing Spezieller Nahrstoffarmer Agar (SNA) medium and incubated for seven days. SNA plates were scraped with sterile distilled water and the spore suspension was evenly distributed onto new SNA plates and incubated under the same above-mentioned conditions. Spore suspension was prepared by scraping 7-days-old SNA plates. The macroconidia were filtered through two layers of cheesecloth to remove mycelial fragments. The concentration of macroconidia suspensions were quantified using a hemocytometer and diluted properly in each assay. Chitosan oligosaccharide sensitivity in vitro Inhibition of mycelial growth Chitosan oligosaccharide (concentration = 95% and N = 5%; pH = 4–6; MO = 80%, molecular weight ≤ 1.500 kDa and water-soluble) was dissolved in 60 mL of sterile distilled water to obtain 200.000 mg/L for the stock solution. The stock solution was added to molten PDA (45–55°C) to obtain the final concentrations of 0 (non-amended agar - PDA), 1000, 2000, 4000, 8000, 16000 and 32000 mg/L. The commercial formulation of tebuconazole (Tebufort 200 g/L, UPL) was used as a control. The tested concentrations of tebuconazole were: 0, 0.5, 1, 2, 4 and 8 mg/L. A mycelial agar plug (5 mm) from the edge of a 7-day-old culture on PDA was placed in the center of a 90-mm-diameter Petri dish containing 15-ml of amended PDA. After 4 days of incubation at 25°C with a 12-h dark/light cycle, radial growth was measured in two perpendicular directions using a digital caliper. Three replicates were used for each concentration and the experiment was repeated once in time. Inhibition of macroconidia germination The tested concentrations for chitosan oligosaccharide were: 0 (non-amended agar - PDA), 62.5, 125, 250, 500, 1000, and 2000 mg/L. The inhibition of macroconidia germination assay was performed using the glass drop technique (Dhingra and Sinclair 1995). Macroconidial suspensions were obtained as described previously with some modifications. SNA plates (7-days-old cultures) were scraped with sterile distilled water with Tween 20 (0.01%) and gelatin (6%). Suspensions were diluted to obtain a final concentration of 1 x 10 7 macroconidia/mL. The commercial formulation of pyraclostrobin (Comet 250 g/L, BASF) was used as a control. The tested concentrations of pyraclostrobin were: 0, 0.01, 0.1 and 1 mg/L. A 30-µL drop of macroconidial suspension was transferred to a glass slide and mixed to a 30-µL drop of chitosan oligosaccharide or pyraclostrobin stock solutions. The glass slides were aligned on a moistened sterile paper towel and placed inside a closed plastic box. The boxes were incubated in the dark at 25°C for 7 hours (Duan et al. 2020 ). Macroconidia germination was assessed by randomly counting fifty macroconidia under a light compound microscope (germinated macroconidia was considered when the germination tube grows to at least half the total length of the conidia). Three replicates (slides) were prepared for each isolate and the experiment was repeated once. Solutions pH and electrical conductivity For the quantification of pH and electrical conductivity, solutions were prepared for each of the doses used in in vitro and plant assays. The levels of these solutions were determined by measuring aliquots with a pH meter for pH values and using the conductivity meter for electrical conductivity (CG2000, GEHAKA, Brazil). All measurements were carried out in triplicate. Chitosan oligosaccharide in seed treatment Wheat seeds (BR18 Terena) were treated with chitosan oligosaccharide (2000 mg/L) and tebuconazole (2.5 mg/L). A sample of 1200 seeds were separated and surface-sterilized (30 s in 70% alcohol, 2 min in 2% sodium hypochlorite, followed by 3 washes in sterile distilled water) to remove the presence of background contaminants that may be present on the seed surface. The seed sample was divided into two sub-samples. The first sub-sample was inoculated by soaking the seeds in a spore suspension (1 x 10 4 macroconidia/mL) for 2 min and allowed to dry on sterile paper towels for 4.5 h. The other subsample was mock-inoculated using the same approach. Half of the inoculated and the mock-inoculated seeds were surface sterilized before seed treatment. After inoculation, 400 seeds (200 inoculated and 200 mock-inoculated) were treated with tebuconazole, and 400 seeds were treated with chitosan oligosaccharide. Treated seeds were immersed into the stock solutions for 5 min and allowed​​ to dry on sterile paper towels for 4 h. The control treatments consisted of 400 seeds, of which 200 were inoculated (100 disinfested and 100 non-disinfested) and 200 non-inoculated (100 disinfested and 100 non-disinfested). Each treatment consisted of 100 seeds divided into five plastic boxes (replicates) with four sheets of germitest paper, previously moistened with sterile saline solution (12 g/L) and containing 20 seeds each. Plastic boxes were incubated at 25°C with a 12-h light/dark cycle for 6 days. The incidence of affected seeds was assessed as the percentage of seeds within each box showing F. graminearum- like growth. The experiment was repeated once. Another assay was performed to investigate the effect of seed treatment in seed germination. For such, the same procedures described above were used with only one modification. The seeds for each treatment were also divided into five plastic boxes with four sheets of germitest paper, previously moistened with sterile water instead of saline solution to allow the seeds to absorb water and to germinate. Plastic boxes were also incubated at 25°C with a 12-h light/dark cycle for 6 days. Five replicates were prepared for each treatment (plastic box) with 20 seeds each. The incidence of affected seeds was assessed as the percentage of seeds within each box showing F. graminearum- like growth. Seed germination was assessed by counting the number of germinated seeds out of the twenty within each plastic box. Seed was considered germinated when the coleoptile was larger than twice the length of the seed. The experiment was repeated once. Chitosan oligosaccharide in the whole plant applications Plant growth conditions Sowing was carried out in a greenhouse at Universidade Federal de Viçosa, MG, Brazil (20°45'29.12" S, 42°52'11.92" W, 660 m above sea level) during the April 2021 - August 2021 and April 2022 - August 2022 harvest. Ten seeds of the FHB-susceptible cultivar BR 18 Terena, were sown in 1-liter pots containing commercial substrate (Sousa 2002 ). After two weeks, the plants were thinned to keep 5 plants/pot and were staked with wire tutors to prevent the plants from tipping over. After that, the plants receive weekly fertilization in the form of a nutrient solution. Each pot received 50 mL of the solution prepared with 6.4mg/L KCl, 3.48mg/L K2SO4, 5.01mg/L MgSO4.7H2O, 2.03mg/L (NH2)2CO, 0.009mg/L NH4MO7O24.4H2O, 0.054mg/L H3BO3, 0.222mg/L ZnSO4.7H2O, 0.058mg/L CuSO4.5H2O, 0.137mg/L MnCl2.4H2O, 0.27g/L FeSO4.7H2O, and 0.37g/L disodium-EDTA prepared with distilled water (Filha et al. 2011 ). Four replicates were prepared for each treatment (pots with five plants each). The experiment was repeated once. Assessments include counting symptomatic spikelets at 8, 11, 13, and 21 days after inoculation. Disease severity (%) was determined as the percentage of the symptomatic spikelets within each wheat head. Plant inoculation procedures Spore suspensions were prepared as described previously. The macroconidia suspension concentration was adjusted to a final concentration of 1 x 10 4 macroconidia/mL. Inoculations were carried out when the plants reached the mid-anthesis stage (Feeks growth stage 10.5.2, the stage of greatest susceptibility to infection by the FHB pathogen (Miller 1999 ; Strange and Smith 1971 ; Strange et al. 1974 ). Plants were spray-inoculated (1 mL/head) using a 0.5 L manual plastic sprayer (Vonder). After the inoculation, the heads were covered with a transparent plastic bag and kept in an incubator at 25°C with a 12-h dark/light cycle for 24 hours. Effect of application timing of chitosan oligosaccharide sprays For this assay, the concentration of 2000 mg/L of chitosan oligosaccharide and 2.5 mg/L of the commercial formulation of tebuconazole were used. The products were sprayed using a 0.5 L manual plastic sprayer (Vonder) at different times, 5 and 2 days before and after inoculation (-5, -2, + 2, +5 days) and on the day of the inoculation one hour before to allow plant to dry out (0 days). Nonsprayed plants and noninoculated plants were used as controls. Effect of concentration of chitosan oligosaccharide For this assay, the concentrations of 500, 1000, 2000 and 3000 mg/L of chitosan oligosaccharide and 2.5 mg/L of the commercial formulation of tebuconazole were used. Plants were sprayed with each concentration individually using a 0.5 L manual plastic sprayer (Vonder), 5 days before inoculation at mid-anthesis. Nonsprayed plants and noninoculated plants were used as controls. Effect as a biostimulant To evaluate whether the chitosan oligosaccharide would induce any biostimulant effect, at the end of the cycle, before harvesting heads, the height of the plants was measured using a tape measure. After harvesting, wheat heads were hand threshed and the grains were weighed, to obtain the thousand-kernel weight (TKW), and visual analysis of these grains was also performed to determine the percentage of Fusarium- damaged kernels (FDK). Field trials The field trial was conducted in the experimental station at Universidade Federal de Viçosa (20°44'48.11" S, 42°50'57.51" W, 679 m above sea level) during the (April - August) in the 2021 growing season. The cultivar BR 18 Terena was also used in this trial. The treatments used in the field trial were 5 and 2 days before and after inoculation (-5, -2, + 2, +5 days) and on the day of the inoculation one hour before to allow the plant to dry out (0 days). After inoculation, wheat heads were covered with a transparent plastic bag for 24 hours. Experimental units consisted of microplots (1 x 1m). Five to six plants were inoculated per plot. Mycotoxins analysis Deoxynivalenol concentrations were determined from wheat kernels harvested from the greenhouse and field trials. Dried wheat heads were harvested at the end of the season, threshed and the kernels were stored at -20°C until analysis. DON were determined by bulking the grains from the individual heads ( n = 5–6 heads per pot) and replicates ( n = 3–4 pots). A 10 g subsample of the pooled kernels was sent to the Laboratory SAMITEC - Soluções Análiticas Microbiológicas e Tecnológicas Ltda, in Santa Maria, for analysis of DON. The amount of DON was quantified in the samples using a gas chromatography-mass spectrometry method as described previously (Mirocha et al. 1998 ; Fuentes et al. 2005 ). Experimental design and data analysis All experiments were conducted as a completely randomized design. Data from the replication of the assays in time were combined for the analysis when the experiment (added as a fixed effect in the model) was not significant. Effective concentration leading to a 50% reduction of mycelial growth or macroconidia germination (EC 50 ) and the respective standard error (SE) was estimated using the 'ec50estimator' and 'drc' packages (Ritz et al. 2015 ; Alves 2020 ). The data of seed treatment and foliar application assays were previously tested for normality and homoscedasticity, and the assumptions were not met, data were transformed using the square root transformation (y' = \(\:\sqrt{y}\) ) and then subjected to the analysis of variance (ANOVA). Thus, the effect of main factors and the interaction between them were evaluated by the F test (α = 5%). Post hoc analyses were performed with the 'emmeans' package to obtain the estimates of the marginal means and the respective confidence intervals (Lenth et al. 2020 ). The ‘cld’ function from ‘multicomp’ R package (Hothorn et al. 2008 ) was used for multiple comparison of treatment means at 5% significance using Tukey test. Whenever there was a significant effect of the interaction between the factors, multiple comparisons were conducted, unfolding all combinations of the levels of each factor within the other factors. All analyses and plots were produced using R software (R Core Team 2025). Results Chitosan oligosaccharide sensitivity in vitro Overall, the mycelial growth and conidia germination of F. graminearum were reduced with the increase of concentrations of tebuconazole and pyraclostrobin, respectively, and also for the chitosan oligosaccharide. For the mycelial growth assays using chitosan oligosaccharide and tebuconazole, the estimated EC 50 values were 7,155.9 mg/L (± 1,344.69 standard error [SE]) and 0.11 mg/L (± 0.05 SE) respectively and for conidia germination assay using chitosan oligosaccharide and pyraclostrobin the estimated EC 50 were 519.31 mg/L (± 28.07 SE) and 0.0002 mg/L (± 0.21 SE), respectively. Solutions pH and electrical conductivity Overall, pH values showed a reduction resulting from the increase in concentration (Table S1). As for electrical conductivity, the effect was the inverse, with increasing concentrations leading to increasing pH values. Chitosan oligosaccharide in seed treatment There was a significant effect for the triple interaction between the factors (treatment, inoculation, and surface sterilization; P ≤ 0.05), but not for the experiment replication as a fixed effect ( P = 0.808). For this reason, data from the two experiments were combined (Table 1). The incidence of F. graminearum in the inoculated and non desinfested control significantly higher than in the other controls (31.5%) ensuring that both the inoculation and the surface sterilization method were effective. The incidence of the pathogen in seeds treated with tebuconazole and chitosan oligosaccharide, disinfested and non disinfested, ranged from 6 to 7.5% and 8.5 to 15.5%, respectively. Tebuconazole and chitosan oligosaccharide treatments were able to reduce the incidence of F. graminearum in the seeds, differing statistically from the control, when the seeds were inoculated but not surface sterilized. On the other hand, there was no significant difference between the treatments when the seeds were non-inoculated, regardless of surface sterilization. Similar results were observed in the assay aiming to investigate the effect of seed treatment in seed germination. There was a significant effect for the triple interaction between the factors (treatment, inoculation, and surface sterilization; P ≤ 0.05), but not for the experiment replication as a fixed effect ( P = 0.465). For this reason, data from the two experiments were also combined (Table 2). The highest incidence of F. graminearum were observed in the inoculated and non-surface sterilized control. Consequently, the highest reduction in the percentage of seed germination was also observed for this treatment, showing a direct association between the presence of the fungus in the seeds and the reduction of the germination. The incidence of the pathogen in seeds treated with tebuconazole and chitosan oligosaccharide disinfested and non disinfested, ranged from 0 to 0.993% and from 1.49 and 6.33%, respectively. Seed germination for these treatments ranged from 65 to 80% and from 62 to 85%, respectively. Greenhouse assay Concentration of chitosan oligosaccharide The AUDPC values in the first and second trials, using different doses of chitosan oligosaccharide, did not differ significantly from the non-treated check (NTC), with mean values ranging from 152.8 to 296.7 (Figure 1 A and B). Even without showing significant differences, a variation in the percentage of AUDPC reduction is apparent in the two assays. There was a significant difference for tebuconazole treatment compared to the NTC in both trials, with the mean AUDPC of 4.13 and 18.8 in the first and second trial, respectively. In both trials, mean AUDPC for chitosan oligosaccharide and for tebuconazole were significantly different from each other with the fungicide performing best. No FHB symptoms were presented in the mock-inoculated plants in both trials. Similarly to the previous trial, DON was detected in all samples of bulked kernels, except in the mock-inoculated control ( data not shown ). Overall, there was an association between the reduction of mean AUDPC (Figure 1 A and B) and reduction in DON accumulation wheat grains (Figure 4). Similarly, some treatments with chitosan oligosaccharide reduced both disease (AUDPC) and suppressed DON accumulation compared to the NTC. The treatments Chi 1000 in the first trial and Chi 500 in the second trial, reduced AUDPC in 36.6 and 20.6% and DON in 47.6 and 42.4% respectively. Consistently, fungicide treatment in both experiments was able to suppress DON accumulation from 78.6 up to 91.4% (Figure 1 C and D). Timing of chitosan oligosaccharide sprays Overall, there was a higher FHB severity in the second trial compared to the first trial (Figure S1). Means values for AUDPC in plants treated with chitosan oligosaccharide ranged from 118.92 to 276.91, with the highest value presented in the treatment chi -2 in the first trial and chi -5 in the second, and for tebuconazole in inoculated plants, mean AUDPC values ranged from 37.20 to 89.60 (Figure 2 A and B). In general, both fungicide and chitosan oligosaccharide performed best in the first trial but only fungicide significantly reduced AUDPC compared to the NTC. No FHB symptoms were presented in the mock-inoculated plants in both trials. DON was detected in all samples of bulked kernels, except in the mock-inoculated control ( data not shown ). There was an association between the reduction of mean AUDPC (Figure 1) and reduction in DON accumulation wheat grains (Figure 2 C and D). In general, some treatments with chitosan oligosaccharide suppressed DON accumulation compared to the NTC. For example, the treatments Chi -5 in the first trial and Chi 0 and Chi +2 in the second trial, with reduction ranged from from 13.6 to 42.3%. Additionally, fungicide treatments were able to suppress DON accumulation from 49 to up to 97.1%. Field assay Overall, there was a large variation in the data which may have prevented us from detecting any significant difference between the treatments. Although not significant, there was a slight reduction in FHB severity on inoculated wheat heads treated with chitosan oligosaccharide or tebuconazole. The highest reductions in disease severity were observed in plants treated with fungicide (44.5 to 100%) regardless of when it was applied. For plants treated with chitosan oligosaccharide, highest reduction in FHB severity was observed in the plants sprayed two days before inoculation (49.70%) followed by treatments applied on the day of inoculation (39.30%). On average, all treatments were able to reduce in relation to the nontreated check, ranging from 12.0 to 100%, except for chitosan oligosaccharide applied five days before inoculation (Figure 3). Biostimulant effect There was no significant effect of chitosan oligosaccharide or even fungicide sprays in increasing treated plants height ( P > 0.05), regardless of the concentration tested (Table S2) or timing of those sprays (Table S3). The percentage of Fusarium -damaged kernels ranged from 0 to 48.5% across all trials. Tebuconazole treatments significantly reduce FDK in inoculated plants across all trials (Table S4 and S5) compared to the NTC, except for the field trial (Table S6). Conversely, chitosan oligosaccharide regardless of the concentration tested (Table S5) or timing of those sprays (Table S4) did not significantly reduce FDK compared to the NTC. Thousand-kernel weight ranged between 25.80 and 53.20% across all trials (Tables S7, S8 and S9). Mean TKW was highest in plants treated with tebuconazole sprayed in different timings (Table S3). For chitosan oligosaccharide, some concentrations and timings were able to prevent TKW reduction of up to 50% under greenhouse conditions. There was no significant reduction in TKW in inoculated plants either sprayed with chitosan oligosaccharide or tebuconazole in the field (Table S9). Discussion In this study, the potential sole use of the chitosan oligosaccharide in FHB management as well as its effect in suppressing DON accumulation in grains was investigated. In the literature, the use of chitosan and its derivatives is well reported for the control of pathogens and diseases. Several studies have investigated the potential use of chitosan in a wide range of plant pathogens, either the direct effect during fungal cultivation in vitro , for example Sclerotium rolfsii in carrots (Ahmed et al. 2019 ), Botrytis cinerea in kiwi (Hua et al. 2019 ), Pseudoperonospora cubensis in cucumber (Henríquez-Díaz et al. 2020 ), Magnaporthe oryzae in rice (Lopez-Moya et al. 2021 ), or to be used in post-harvest treatment of Colletotrichum gloeosporioides, Colletotrichum acutatum, Fusarium oxysporum and Phytophthora sp. in strawberry ((Melo et al. 2020 ), Penicillium citrinum and Penicillium mallochii in orange (Coutinho et al. 2020 ), Lasiodiplodia theobromae and Colletotrichum gloeosporioides avocado (Obianom et al. 2019 ), and others. The incidence of F. graminearum in seeds treated with tebuconazole and chitosan oligosaccharide, disinfested and not disinfested, were consistently reduced. The effect of chitosan oligosaccharide on the reduction of F graminearum incidence may be caused by a direct effect on the pathogen infecting seed surface. The direct effect of chitosan in pathogens infecting plant tissue surfaces have already been reported in other pathosystems, for example on gray mold and blue mold caused by Botrytis cinerea and Penicillium expansum in tomato fruit (Liu et al. 2007 ). More studies are needed to investigate the potential of oligosaccharide to reduce the incidence of already established infections, either by direct effect or inducing plant resistance. In addition to controlling the pathogen, the chitosan oligosaccharide did not interfere with seed germination and, even if not significant, the chitosan oligosaccharide showed a slight increase in seed germination. Some studies in the literature mention that compounds derived from chitosan present can increase seed germination in several crops. Zohara et al. ( 2019 ) tested the use of chitosan as a biostimulant to control infection of cucumber by Phytophthora capsici and observed that in addition to controlling the pathogens, chitosan improve the germination rate and some traits such as shoot and root weight. Mazaro et al. ( 2009 ) evaluated the effect of treating sugar-beet and tomato seeds with chitosan to control of damping-off in substrate infested with Rhizoctonia sp., as a result, they reported that there was a reduction in the incidence of the pathogen, reducing the number of plants felled, which may be related to the increase in phenylalanine ammonia-lyase, since it acts in the formation of many plant defense mechanisms. Further studies can be conducted to better understand the effect of the seed treatment with chitosan oligosaccharide in the development of the wheat plants. In the present study, the effect of chitosan oligosaccharide directly on F. graminearum was investigated. Although the estimated EC 50 values were high compared to the fungicides included as controls, the tested isolate was sensitive to chitosan oligosaccharide as concentration was increased. These findings show that chitosan oligosaccharide can directly reduce both mycelial growth and macroconidia germination of F. graminearum . The direct effect of chitosan in some pathogens, interfering with mycelial growth, have already been reported (Berger et al. 2011 ). Previous studies have already reported the effect of chitosan inhibiting F. graminearum mycelial growth and conidia germination in vitro (Kheiri et al. 2016 ) and the effect of oligochitosan and chitosan hydrochloride, other chitosan derivatives, to inhibit mycelial growth for F. graminearum (Xu et al. 2007 ; Deshaies et al. 2022 ) showing that as the concentration increased, the growth of the pathogen was affected. Increasing the chitosan oligosaccharide doses led to a reduction in pH values and an increase in electrical conductivity values. Sporangia and zoospores of Phytophthora ramorum were tested in response to different electrical conductivity values. The authors showed that in conditions where the values of electrical conductivity and pH were high there was a reduction in the viability of P. ramorum zoospores (Kong et al. 2012 ). Another study on the use of biochar showed that the use of this compound in the soil significantly increased the soil pH, electrical conductivity and other parameters. Furthermore, an increase in bacteria that promote biocontrol, such as Bacillus subtilis and suppression of pathogens of the genera Fusarium and Ilyonectria , was observed (Wang et al. 2020 ). In the greenhouse experiments, chitosan oligosaccharide was sprayed in different timings around anthesis when the inoculation was performed. Kheiri et al.(2016) applied chitosan on wheat plants in 0, 3 and 5 days by spraying after inoculation. They observed that there was a reduction in severity of around 30% in treatments with chitosan about the control also failing to differentiate the influence of the application period on disease control. Deshaies et al. ( 2022 ) observed that even when chitosan was applied pre or post inoculation in wheat plants for F. graminearum , the control was effective, reducing AUDPC. Unfortunately, our data did not provide enough evidence to confirm this hypothesis. Although not significantly different from the NTC, there was a reduction in FHB severity and AUDPC in the plants sprayed five days before inoculation in the first trial and on the day of the inoculation in the second trial. These inconsistencies between them may be explained, at least in part, by weather conditions during each trial (data not shown). The disease progress rate was slower in the nontreated check in the first trial compared with the second, when the conditions were more conducive for the development of the disease. Further studies are needed to understand the best time of application of chitosan oligosaccharide to control FHB in wheat. Our results suggest that some treatments were effective for the reduction of deoxynivalenol. In the first trial, in the treatment Chi − 5 there was a reduction of 33.1% and in the second, in the treatment Chi 0 of 42.3% and + 2 of 13.6% in the treatments that were applied to the chitosan oligosaccharide. Silva et al. (2015) used a chitosan film to reduce the aflatoxin produced by Aspergillus parasiticus in peanuts. The authors reported that the reduction of aflatoxin in the grains was greater than 80%. Zachetti et al. ( 2019 ) tested the use of chitosan as an alternative to control the growth and mycotoxin of Fusarium verticilioides, F. proliferatum (fumonisin) and F. graminearum (deoxynivalenol) production in corn and wheat and showed that chitosan had an effect on the reduction of these mycotoxins, as demonstrated in this study. Chitosan oligosaccharide did not increase control efficacy with the increase of the concentration sprayed in wheat plants. The plants treated with different doses of chitosan oligosaccharide, applied five days before inoculation some doses were able to reduce the severity of F. graminearum in relation to the control, in the different days of evaluation and consequently reduce the AUDPC, but there were no significant differences between them. Zachetti et al. ( 2019 ) conducted an in vitro study using different doses of chitosan to inhibit the mycelial growth of different Fusarium species. In their results, for Fusarium graminearum , they observed that, regardless of the dose used, there was no interference in the growth of the isolate. In our study, as discussed above, in the in vitro tests we were able to observe a decrease in mycelial growth and macroconidia germination with the increase of chitosan oligosaccharide concentrations, which were not observed in planta . Additionally, the application of chitosan oligosaccharide did not result in increased plant height in all assays. Cancio et al. ( 2014 ) included in their study that chitosan-treated bean seeds did not show significant differences in plant height when compared to the control. The weight of 1000 grains was also evaluated. The treatments with chitosan oligosaccharide applied 5 days before inoculation showed an increase of 11.8% in the final weight of the grains. In the second trial, the treatments applied on the day of inoculation and 2 days after inoculation showed an increase of 14.76 and 7.05%. He et al. ( 2018 ) using chitosan oligosaccharide for pre-harvest treatment of strawberry and concluded that the product brought an increase in viscosity, lignin, sugar, protein, total soluble solids and titratable acidity in the fruits. In conclusion, in the in vitro test, chitosan oligosaccharide was able to reduce mycelial growth and conidia germination at higher doses of the product. In the greenhouse and field trials, chitosan oligosaccharide was able to reduce the FHB intensity, although it did not differ from the control. The amount of deoxynivalenol accumulated in wheat grains inoculated under greenhouse conditions was reduced in some greenhouse treatments. Our results suggest that chitosan oligosaccharide has some direct effect on F. graminearum , but further studies are needed to better understand the in vivo efficacy of this product in combination with fungicides. Declarations Authors’ contributions Conceptualization: G.F.P. and F.J.M.; methodology: G.F.P., F.J.M. and E.M.D.; investigation: G.F.P., L.L.S.A., R.A.S., J.M.S and F.J.M.; statistical analysis: G.F.P., F.J.M. and E.M.D.; writing - original draft preparation: G.F.P. and F.J.M.; writing - review and editing: G.F.P., L.L.S.A., R.A.S., J.M.S., F.J.M. and E.M.D.; supervision: F.J.M. All authors have read and agreed to the published version of the manuscript. Data availability statement The data and R codes used in the analysis are available as a research compendium in https://github.com/Gabrielgt40/Analysis-paper-COS01.git Acknowledgements We thank the Programa de Pós-graduação em Fitopatologia (UFV) and Capes/Proex (Programa de Aperfeiçoamento de Pessoal de Nível Superior/Programa de Excelência Acadêmica) for providing a graduate scholarship to G. F. Paiva. We thank the ALAS (América Latina Agricultura Sustentável) for the financial support. Conflict of interest statement On behalf of all authors, the corresponding author states that there is no conflict of interest. Funding This study was partially funded by Capes/Proex (Programa de Aperfeiçoamento de Pessoal de Nível Superior/Programa de Excelência Acadêmica) and ALAS (América Latina Agricultura Sustentável). Graduate scholarship of G. F. Paiva was provided by CAPES. References Ahmed MU, Bhuiyan MKA, Hossain MM, et al (2019) AND IMPROVEMENT THE CROP PRODUCTION. Vet Sci 13 Almeida RS, Peniche CA, Solís Y, et al (2019) Produção, caracterização e avaliação in vitro de partículas de quitosana e hidroxiapatita para substituição óssea. 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Treatments Surface sterilization Inoculation Tebuconazole Chitosan oligosaccharide Check Disinfested Inoculated 7.5 Aa 18 Bb* 13.5 ABa Non disinfested 6 Aa 10 Aa 31.5 Bb* Disinfested Not inoculated 6.5 Aa 8.5 Aa 12 Aa Non disinfested 7.5 Aa 15.5 Aa 13.5 Aa The means followed by the lowercase letters in the columns and not followed by an asterisk in the columns did not differ from each other at 5% of significance by Tukey test. Table 2. Combined data from the two experiments, estimating the mean values of F. graminearum incidence in the germination assay in wheat seeds. Incidence (%) Seed germination (%) Surface sterilized Inoculation Tebuconazole Chitosan oligosaccharide Check Tebuconazole Chitosan oligosaccharide Check Disinfested Inoculated 0.99 Aa 1.49 Aa 3.98 Aa 65 Aa 74 Aa 69 Ab Non disinfested 0 Aa 6.33 Bb 28.52 Cb 77 Bb 77 Ba 55 Aa Disinfested Not inoculated 0.46 Aa 3.26 Aa 0.30 Aa* 80 Aa 72 Aa 80 Aa* Non disinfested 0.10 Aa 2.27 Aa 0.46 Aa* 80 Aa 85 Ab 81 Aa* The means followed by the lowercase letters in the columns and not followed by an asterisk in the columns did not differ from each other at 5% of significance by Tukey test. 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09:27:43","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":178150,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7915619/v1/2c9fe12e0f9c3a933191b1f8.html"},{"id":96707024,"identity":"ca82e771-6b09-4286-b891-a8103a240e62","added_by":"auto","created_at":"2025-11-25 09:27:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":96187,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Estimated mean area under disease progress curve (AUDPC) and respective 95% confidence intervals of Fusarium head blight severity in wheat heads sprayed at different concentrations in the first and (B) second trial. (C) Deoxynivalenol (DON) production in bulked kernel samples from inoculated wheat heads sprayed with different concentrations of chitosan oligosaccharide in the first and (D) second trial. NTC = Nontreated check, Chi 500 = 500 mg/L chitosan oligosaccharide, Chi 1000 = 1000 mg/L chitosan oligosaccharide, Chi 2000 = 2000 mg/L chitosan oligosaccharide, Chi 3000 = 3000 mg/L chitosan oligosaccharide, Tebuc 2.5 = 2.5 mg/L tebuconazole. Means followed by the same uppercase letters did not differ from each other at 5% significance by Tukey test.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7915619/v1/6e76618d9bcbf4e8a588be1c.png"},{"id":96711121,"identity":"77acafa0-39f4-4a8a-970c-9faeac7e840c","added_by":"auto","created_at":"2025-11-25 10:11:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":117522,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Estimated mean area under disease progress curve (AUDPC) and respective 95% confidence intervals of Fusarium head blight severity in wheat heads sprayed at different timings in the first and (B) second trial. (C) Deoxynivalenol (DON) production in bulked kernel samples from inoculated wheat heads sprayed with different concentrations of chitosan oligosaccharide in the first and (D) second trial. Treatments: NTC = Nontreated check, Chi = chitosan oligosaccharide and Tebuc = tebuconazole. Chi or Tebuc followed by: -5 = applied 5 days before inoculation, -2 = applied 2 days before inoculation, 0 = chitosan oligosaccharide applied on the day of inoculation, and +2 applied 2 days after inoculation. NTC = Nontreated check. The values below the points represent the percentage reduction compared to the NTC. Means followed by the same uppercase letters did not differ from each other at 5% significance by Tukey test.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7915619/v1/df04c01cff772478529c4366.png"},{"id":96707020,"identity":"ccd27678-2e8e-46b2-8d63-c1904f63dced","added_by":"auto","created_at":"2025-11-25 09:27:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":58720,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Mean Fusarium head blight severity and respective 95% confidence intervals in wheat heads 21 days after inoculation sprayed at different timings in the first and (B) second trial. Treatments: NTC = Nontreated check, Chi = chitosan oligosaccharide and Tebuc = tebuconazole. Chi or Tebuc followed by: -5 = applied 5 days before inoculation, -2 = applied 2 days before inoculation, 0 = chitosan oligosaccharide applied on the day of inoculation, and +2 applied 2 days after inoculation. Means followed by the same uppercase letters did not differ from each other at 5% significance by Tukey test.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7915619/v1/cfc88b2927ad6c2be2859f16.png"},{"id":97135550,"identity":"c469ffce-2271-4628-a5fd-93b3e6e29c88","added_by":"auto","created_at":"2025-12-01 09:50:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1286989,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7915619/v1/a725c0ae-b135-47cd-a912-08dfb8db6bce.pdf"},{"id":96707023,"identity":"8c6378fe-cf39-4dd2-ab49-3f162eef775a","added_by":"auto","created_at":"2025-11-25 09:27:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1896995,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYMATERIAL.docx","url":"https://assets-eu.researchsquare.com/files/rs-7915619/v1/7ae744025a88b221a454cf49.docx"}],"financialInterests":"","formattedTitle":"Evaluation of chitosan oligosaccharide as an alternative treatment to mitigate Fusarium graminearum and mycotoxin contamination in wheat","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFusarium head blight (FHB), caused by members of the \u003cem\u003eFusarium graminearum\u003c/em\u003e species complex, among which \u003cem\u003eFusarium graminearum\u003c/em\u003e s.s. is one of the most dominant species, is one of the important diseases of wheat worldwide. Losses due to FHB in wheat and barley crops in the United States is estimated at around \u003cspan\u003e$\u003c/span\u003e2.7\u0026nbsp;billion. The disease has been considered a resurgent problem worldwide since the early 1990s, posing significant challenges to agricultural production and food security, and grain quality due to the accumulation of mycotoxins, such as the B-trichothecene deoxynivalenol (DON), nivalenol (NIV) and their acetyl-derivatives (Duffeck et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Figueroa et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; McMullen et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Del Ponte et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Nganje et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Tralamazza et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). FHB is considered a floral and monocyclic disease, where primary infection occurs from inoculum surviving in plant debris. The main symptoms are bleaching of spikelets with the presence of pinkish or orange-ish signs of the pathogen in the base of the glumes, leading to kernel infection, yield reduction, and contamination of tissues and grains with mycotoxins (Snijders \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Gilbert and Haber \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The resurgence of FHB s, in southern Brazil, a subtropical environment, has been associated mainly with climate variability, while the role of within-field local inoculum has been questioned, which contrasts with studies in the temperate regions climatic factors (Markell and Francl \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Del Ponte et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Duffeck et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe management of FHB aims to reduce disease intensity as well as mycotoxin levels to meet the maximum tolerated limits in wheat products as established elsewhere for each country, including Brazil (ANVISA 2011, 2017, 2022). There are several methods available for the management of FHB, including cultural practices, resistant cultivars, chemical control, biological control, and others, with the the integration of all these practices being more more effective than any one applied in isolated (Wegulo et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Chemical control is one of the most used measures for an effective FHB control. Demethylation inhibitor fungicides (DMI) are the most widely used for the control of disease, and the reduction in the accumulation of mycotoxins (Paul et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; McMullen et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wegulo et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Overall, the fungicides used to control FHB show variable efficacy. In a meta-analytic study in North America, it has been shown a large variation of control efficacy across the trials, with performances ranging from 32 to 50% for different the DMI fungicides tebuconazole, propiconazole, metconazole, and prothioconazole (Paul et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and in Brazil for triazole and benzimidazole (Machado et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). With the recurrent use of fungicides in the management of this disease, there is a risk to select resistant individuals within the \u003cem\u003eF. graminearum\u003c/em\u003e population. In fact, in the state of New York and in Henan Province in China, tebuconazole less sensitivity isolates of \u003cem\u003eF. graminearum\u003c/em\u003e have been reported (Spolti et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, new control methods must be developed.\u003c/p\u003e\u003cp\u003eThe use of other products such as chitosan derivatives may be a viable option to integrate management targeting disease and mycotoxin control. Chitosan is a polymer of D-glucosamine derived from the deacetylation of chitin which is found naturally in the cell wall of fungi and also in the exoskeleton of crustaceans, being considered the second most abundant polysaccharide in nature (Berger et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Almeida et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Chitosan has been studied for its ability to increase plant tolerance to stress and to activate plant defense responses (Katiyar et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Suarez-Fernandez et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Saberi Riseh et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Previous studies have already reported the effect of chitosan inhibiting \u003cem\u003eF. graminearum\u003c/em\u003e mycelial growth and conidia germination in vitro (Kheiri et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Deshaies et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eChitosan oligosaccharides are the degraded products of chitin or chitosan by acid hydrolysis and/or enzymatic degradation, and it is considered a chitosan oligomer, and compared to chitosan, has a higher water solubility and lower viscosity (Yin et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Muanprasat and Chatsudthipong \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Naveed et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The chitosan oligosaccharide, after applied to the plant surface, is recognized by a receptor on the plant cell membrane, followed by signal transfer and amplification, activation of responsive genes, accumulation of responsive proteins, induction of defense-related secondary metabolites, and finally the plant response, such as hypersensitivity responses, production of phytoalexins and reinforcement of cell walls (Yin et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This study aims to investigate the potential of chitosan oligosaccharide, a chitosan derivative, as an alternative control product, sprayed alone, for the control of FHB intensity as well as DON accumulation in wheat grains.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eFungal isolate and inoculum preparation\u003c/h2\u003e\u003cp\u003eA \u003cem\u003eF. graminearum\u003c/em\u003e DON/15ADON-producing isolate (CML 3066), representative of the dominant population in Southern Brazil, was chosen for this study (Del Ponte et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wood et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The isolate was grown on potato dextrose agar (PDA) at 25\u0026deg;C with a 12-h dark/light cycle for seven days. Afterwards, mycelial plugs were transferred to plates containing \u003cem\u003eSpezieller Nahrstoffarmer\u003c/em\u003e Agar (SNA) medium and incubated for seven days. SNA plates were scraped with sterile distilled water and the spore suspension was evenly distributed onto new SNA plates and incubated under the same above-mentioned conditions. Spore suspension was prepared by scraping 7-days-old SNA plates. The macroconidia were filtered through two layers of cheesecloth to remove mycelial fragments. The concentration of macroconidia suspensions were quantified using a hemocytometer and diluted properly in each assay.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChitosan oligosaccharide sensitivity\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eInhibition of mycelial growth\u003c/h3\u003e\n\u003cp\u003eChitosan oligosaccharide (concentration\u0026thinsp;=\u0026thinsp;95% and N\u0026thinsp;=\u0026thinsp;5%; pH\u0026thinsp;=\u0026thinsp;4\u0026ndash;6; MO\u0026thinsp;=\u0026thinsp;80%, molecular weight\u0026thinsp;\u0026le;\u0026thinsp;1.500 kDa and water-soluble) was dissolved in 60 mL of sterile distilled water to obtain 200.000 mg/L for the stock solution. The stock solution was added to molten PDA (45\u0026ndash;55\u0026deg;C) to obtain the final concentrations of 0 (non-amended agar - PDA), 1000, 2000, 4000, 8000, 16000 and 32000 mg/L. The commercial formulation of tebuconazole (Tebufort 200 g/L, UPL) was used as a control. The tested concentrations of tebuconazole were: 0, 0.5, 1, 2, 4 and 8 mg/L. A mycelial agar plug (5 mm) from the edge of a 7-day-old culture on PDA was placed in the center of a 90-mm-diameter Petri dish containing 15-ml of amended PDA. After 4 days of incubation at 25\u0026deg;C with a 12-h dark/light cycle, radial growth was measured in two perpendicular directions using a digital caliper. Three replicates were used for each concentration and the experiment was repeated once in time.\u003c/p\u003e\n\u003ch3\u003eInhibition of macroconidia germination\u003c/h3\u003e\n\u003cp\u003eThe tested concentrations for chitosan oligosaccharide were: 0 (non-amended agar - PDA), 62.5, 125, 250, 500, 1000, and 2000 mg/L. The inhibition of macroconidia germination assay was performed using the glass drop technique (Dhingra and Sinclair 1995). Macroconidial suspensions were obtained as described previously with some modifications. SNA plates (7-days-old cultures) were scraped with sterile distilled water with Tween 20 (0.01%) and gelatin (6%). Suspensions were diluted to obtain a final concentration of 1 x 10\u003csup\u003e7\u003c/sup\u003e macroconidia/mL. The commercial formulation of pyraclostrobin (Comet 250 g/L, BASF) was used as a control. The tested concentrations of pyraclostrobin were: 0, 0.01, 0.1 and 1 mg/L. A 30-\u0026micro;L drop of macroconidial suspension was transferred to a glass slide and mixed to a 30-\u0026micro;L drop of chitosan oligosaccharide or pyraclostrobin stock solutions. The glass slides were aligned on a moistened sterile paper towel and placed inside a closed plastic box. The boxes were incubated in the dark at 25\u0026deg;C for 7 hours (Duan et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Macroconidia germination was assessed by randomly counting fifty macroconidia under a light compound microscope (germinated macroconidia was considered when the germination tube grows to at least half the total length of the conidia). Three replicates (slides) were prepared for each isolate and the experiment was repeated once.\u003c/p\u003e\n\u003ch3\u003eSolutions pH and electrical conductivity\u003c/h3\u003e\n\u003cp\u003eFor the quantification of pH and electrical conductivity, solutions were prepared for each of the doses used in \u003cem\u003ein vitro\u003c/em\u003e and plant assays. The levels of these solutions were determined by measuring aliquots with a pH meter for pH values and using the conductivity meter for electrical conductivity (CG2000, GEHAKA, Brazil). All measurements were carried out in triplicate.\u003c/p\u003e\n\u003ch3\u003eChitosan oligosaccharide in seed treatment\u003c/h3\u003e\n\u003cp\u003eWheat seeds (BR18 Terena) were treated with chitosan oligosaccharide (2000 mg/L) and tebuconazole (2.5 mg/L). A sample of 1200 seeds were separated and surface-sterilized (30 s in 70% alcohol, 2 min in 2% sodium hypochlorite, followed by 3 washes in sterile distilled water) to remove the presence of background contaminants that may be present on the seed surface. The seed sample was divided into two sub-samples. The first sub-sample was inoculated by soaking the seeds in a spore suspension (1 x 10\u003csup\u003e4\u003c/sup\u003e macroconidia/mL) for 2 min and allowed to dry on sterile paper towels for 4.5 h. The other subsample was mock-inoculated using the same approach. Half of the inoculated and the mock-inoculated seeds were surface sterilized before seed treatment. After inoculation, 400 seeds (200 inoculated and 200 mock-inoculated) were treated with tebuconazole, and 400 seeds were treated with chitosan oligosaccharide. Treated seeds were immersed into the stock solutions for 5 min and allowed​​ to dry on sterile paper towels for 4 h. The control treatments consisted of 400 seeds, of which 200 were inoculated (100 disinfested and 100 non-disinfested) and 200 non-inoculated (100 disinfested and 100 non-disinfested).\u003c/p\u003e\u003cp\u003eEach treatment consisted of 100 seeds divided into five plastic boxes (replicates) with four sheets of germitest paper, previously moistened with sterile saline solution (12 g/L) and containing 20 seeds each. Plastic boxes were incubated at 25\u0026deg;C with a 12-h light/dark cycle for 6 days. The incidence of affected seeds was assessed as the percentage of seeds within each box showing \u003cem\u003eF. graminearum-\u003c/em\u003elike growth. The experiment was repeated once.\u003c/p\u003e\u003cp\u003eAnother assay was performed to investigate the effect of seed treatment in seed germination. For such, the same procedures described above were used with only one modification. The seeds for each treatment were also divided into five plastic boxes with four sheets of germitest paper, previously moistened with sterile water instead of saline solution to allow the seeds to absorb water and to germinate. Plastic boxes were also incubated at 25\u0026deg;C with a 12-h light/dark cycle for 6 days. Five replicates were prepared for each treatment (plastic box) with 20 seeds each. The incidence of affected seeds was assessed as the percentage of seeds within each box showing \u003cem\u003eF. graminearum-\u003c/em\u003elike growth. Seed germination was assessed by counting the number of germinated seeds out of the twenty within each plastic box. Seed was considered germinated when the coleoptile was larger than twice the length of the seed. The experiment was repeated once.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eChitosan oligosaccharide in the whole plant applications\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003ePlant growth conditions\u003c/h2\u003e\u003cp\u003eSowing was carried out in a greenhouse at Universidade Federal de Vi\u0026ccedil;osa, MG, Brazil (20\u0026deg;45'29.12\" S, 42\u0026deg;52'11.92\" W, 660 m above sea level) during the April 2021 - August 2021 and April 2022 - August 2022 harvest. Ten seeds of the FHB-susceptible cultivar BR 18 Terena, were sown in 1-liter pots containing commercial substrate (Sousa \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). After two weeks, the plants were thinned to keep 5 plants/pot and were staked with wire tutors to prevent the plants from tipping over. After that, the plants receive weekly fertilization in the form of a nutrient solution. Each pot received 50 mL of the solution prepared with 6.4mg/L KCl, 3.48mg/L K2SO4, 5.01mg/L MgSO4.7H2O, 2.03mg/L (NH2)2CO, 0.009mg/L NH4MO7O24.4H2O, 0.054mg/L H3BO3, 0.222mg/L ZnSO4.7H2O, 0.058mg/L CuSO4.5H2O, 0.137mg/L MnCl2.4H2O, 0.27g/L FeSO4.7H2O, and 0.37g/L disodium-EDTA prepared with distilled water (Filha et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Four replicates were prepared for each treatment (pots with five plants each). The experiment was repeated once. Assessments include counting symptomatic spikelets at 8, 11, 13, and 21 days after inoculation. Disease severity (%) was determined as the percentage of the symptomatic spikelets within each wheat head.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003ePlant inoculation procedures\u003c/h3\u003e\n\u003cp\u003eSpore suspensions were prepared as described previously. The macroconidia suspension concentration was adjusted to a final concentration of 1 x 10\u003csup\u003e4\u003c/sup\u003e macroconidia/mL. Inoculations were carried out when the plants reached the mid-anthesis stage (Feeks growth stage 10.5.2, the stage of greatest susceptibility to infection by the FHB pathogen (Miller \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Strange and Smith \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1971\u003c/span\u003e; Strange et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1974\u003c/span\u003e). Plants were spray-inoculated (1 mL/head) using a 0.5 L manual plastic sprayer (Vonder). After the inoculation, the heads were covered with a transparent plastic bag and kept in an incubator at 25\u0026deg;C with a 12-h dark/light cycle for 24 hours.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eEffect of application timing of chitosan oligosaccharide sprays\u003c/h2\u003e\u003cp\u003eFor this assay, the concentration of 2000 mg/L of chitosan oligosaccharide and 2.5 mg/L of the commercial formulation of tebuconazole were used. The products were sprayed using a 0.5 L manual plastic sprayer (Vonder) at different times, 5 and 2 days before and after inoculation (-5, -2, +\u0026thinsp;2, +5 days) and on the day of the inoculation one hour before to allow plant to dry out (0 days). Nonsprayed plants and noninoculated plants were used as controls.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eEffect of concentration of chitosan oligosaccharide\u003c/h2\u003e\u003cp\u003eFor this assay, the concentrations of 500, 1000, 2000 and 3000 mg/L of chitosan oligosaccharide and 2.5 mg/L of the commercial formulation of tebuconazole were used. Plants were sprayed with each concentration individually using a 0.5 L manual plastic sprayer (Vonder), 5 days before inoculation at mid-anthesis. Nonsprayed plants and noninoculated plants were used as controls.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eEffect as a biostimulant\u003c/h2\u003e\u003cp\u003eTo evaluate whether the chitosan oligosaccharide would induce any biostimulant effect, at the end of the cycle, before harvesting heads, the height of the plants was measured using a tape measure. After harvesting, wheat heads were hand threshed and the grains were weighed, to obtain the thousand-kernel weight (TKW), and visual analysis of these grains was also performed to determine the percentage of \u003cem\u003eFusarium-\u003c/em\u003edamaged kernels (FDK).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eField trials\u003c/h2\u003e\u003cp\u003eThe field trial was conducted in the experimental station at Universidade Federal de Vi\u0026ccedil;osa (20\u0026deg;44'48.11\" S, 42\u0026deg;50'57.51\" W, 679 m above sea level) during the (April - August) in the 2021 growing season. The cultivar BR 18 Terena was also used in this trial. The treatments used in the field trial were 5 and 2 days before and after inoculation (-5, -2, +\u0026thinsp;2, +5 days) and on the day of the inoculation one hour before to allow the plant to dry out (0 days). After inoculation, wheat heads were covered with a transparent plastic bag for 24 hours. Experimental units consisted of microplots (1 x 1m). Five to six plants were inoculated per plot.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eMycotoxins analysis\u003c/h2\u003e\u003cp\u003eDeoxynivalenol concentrations were determined from wheat kernels harvested from the greenhouse and field trials. Dried wheat heads were harvested at the end of the season, threshed and the kernels were stored at -20\u0026deg;C until analysis. DON were determined by bulking the grains from the individual heads (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5\u0026ndash;6 heads per pot) and replicates (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3\u0026ndash;4 pots). A 10 g subsample of the pooled kernels was sent to the Laboratory SAMITEC - Solu\u0026ccedil;\u0026otilde;es An\u0026aacute;liticas Microbiol\u0026oacute;gicas e Tecnol\u0026oacute;gicas Ltda, in Santa Maria, for analysis of DON. The amount of DON was quantified in the samples using a gas chromatography-mass spectrometry method as described previously (Mirocha et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Fuentes et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eExperimental design and data analysis\u003c/h2\u003e\u003cp\u003eAll experiments were conducted as a completely randomized design. Data from the replication of the assays in time were combined for the analysis when the experiment (added as a fixed effect in the model) was not significant. Effective concentration leading to a 50% reduction of mycelial growth or macroconidia germination (EC\u003csub\u003e50\u003c/sub\u003e) and the respective standard error (SE) was estimated using the 'ec50estimator' and 'drc' packages (Ritz et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Alves \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The data of seed treatment and foliar application assays were previously tested for normality and homoscedasticity, and the assumptions were not met, data were transformed using the square root transformation (y' = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\sqrt{y}\\)\u003c/span\u003e\u003c/span\u003e) and then subjected to the analysis of variance (ANOVA). Thus, the effect of main factors and the interaction between them were evaluated by the \u003cem\u003eF\u003c/em\u003e test (α\u0026thinsp;=\u0026thinsp;5%). Post hoc analyses were performed with the 'emmeans' package to obtain the estimates of the marginal means and the respective confidence intervals (Lenth et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The \u0026lsquo;cld\u0026rsquo; function from \u0026lsquo;multicomp\u0026rsquo; R package (Hothorn et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) was used for multiple comparison of treatment means at 5% significance using Tukey test. Whenever there was a significant effect of the interaction between the factors, multiple comparisons were conducted, unfolding all combinations of the levels of each factor within the other factors. All analyses and plots were produced using R software (R Core Team 2025).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003ch2\u003e\u003cstrong\u003eChitosan oligosaccharide sensitivity \u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Overall, the mycelial growth and conidia germination of \u003cem\u003eF. graminearum\u003c/em\u003e were reduced with the increase of concentrations of tebuconazole and pyraclostrobin, respectively, and also for the chitosan oligosaccharide. For the mycelial growth assays using chitosan oligosaccharide and tebuconazole, the estimated EC\u003csub\u003e50\u003c/sub\u003e values were 7,155.9 mg/L (\u0026plusmn; 1,344.69 standard error [SE]) and 0.11 mg/L (\u0026plusmn; 0.05 SE) respectively and for conidia germination assay using chitosan oligosaccharide and pyraclostrobin the estimated EC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003ewere 519.31 mg/L (\u0026plusmn; 28.07 SE) and 0.0002 mg/L (\u0026plusmn; 0.21 SE), respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSolutions pH and electrical conductivity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Overall, pH values showed a reduction resulting from the increase in concentration (Table S1). As for electrical conductivity, the effect was \u0026nbsp;the inverse, \u0026nbsp; with increasing concentrations leading to increasing pH values.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eChitosan oligosaccharide in seed treatment\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThere was a significant effect for the triple interaction between the factors (treatment, inoculation, and surface sterilization; \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026le; 0.05), but not for the experiment replication as a fixed effect (\u003cem\u003eP\u003c/em\u003e = 0.808). For this reason, data from the two experiments were combined (Table 1). The incidence of \u003cem\u003eF. graminearum\u0026nbsp;\u003c/em\u003ein the inoculated and non desinfested control significantly higher than in the other controls (31.5%) ensuring that both the inoculation and the surface sterilization method were effective. The incidence of the pathogen in seeds treated with tebuconazole and chitosan oligosaccharide, disinfested and non disinfested, ranged from 6 to 7.5% and 8.5 to 15.5%, respectively. Tebuconazole and chitosan oligosaccharide treatments were able to reduce the incidence of \u003cem\u003eF. graminearum\u0026nbsp;\u003c/em\u003ein the seeds, differing statistically from the control, when the seeds were inoculated but not surface sterilized. On the other hand, there was no significant difference between the treatments when the seeds were non-inoculated, regardless of surface sterilization.\u003c/p\u003e\n\u003cp\u003eSimilar results were observed in the assay aiming to investigate the effect of seed treatment in seed germination. There was a significant effect for the triple interaction between the factors (treatment, inoculation, and surface sterilization; \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026le; 0.05), but not for the experiment replication as a fixed effect (\u003cem\u003eP\u003c/em\u003e = 0.465). For this reason, data from the two experiments were also combined (Table 2). The highest incidence of \u003cem\u003eF. graminearum\u0026nbsp;\u003c/em\u003ewere observed in the inoculated and non-surface sterilized control. Consequently, the highest reduction in the percentage of seed germination was also observed for this treatment, showing a direct association between the presence of the fungus in the seeds and the reduction of the germination. The incidence of the pathogen in seeds treated with tebuconazole and chitosan oligosaccharide disinfested and non disinfested, ranged from 0 to 0.993% and from 1.49 and 6.33%, respectively. Seed germination for these treatments ranged from 65 to 80% and from 62 to 85%, respectively.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eGreenhouse assay\u003c/strong\u003e\u003c/h2\u003e\n\u003ch2\u003e\u003cstrong\u003eConcentration of chitosan oligosaccharide\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; The AUDPC values in the first and second trials, using different doses of chitosan oligosaccharide, did not differ significantly from the non-treated check (NTC), with mean values ranging from 152.8 to 296.7 (Figure 1 A and B). Even without showing significant differences, a variation in the percentage of AUDPC reduction is apparent \u0026nbsp;in the two assays. There was a significant difference for tebuconazole treatment compared to the NTC in both trials, with the mean AUDPC of 4.13 and 18.8 in the first and second trial, respectively. In both trials, mean AUDPC for chitosan oligosaccharide and for tebuconazole were significantly different from each other with the fungicide performing best. No FHB symptoms were presented in the mock-inoculated plants in both trials.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Similarly to the previous trial, DON was detected in all samples of bulked kernels, except in the mock-inoculated control (\u003cem\u003edata not shown\u003c/em\u003e). Overall, there was an association between the reduction of mean AUDPC (Figure 1 A and B) and reduction in DON accumulation wheat grains (Figure 4). Similarly, some treatments with chitosan oligosaccharide reduced both disease (AUDPC) and suppressed DON accumulation compared to the NTC. The treatments Chi 1000 in the first trial and Chi 500 in the second trial, reduced AUDPC in 36.6 and 20.6% and DON in 47.6 and 42.4% respectively. Consistently, fungicide treatment in both experiments was able to suppress DON accumulation from 78.6 up to 91.4% (Figure 1 C and D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTiming of chitosan oligosaccharide sprays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Overall, there was a higher FHB severity in the second trial compared to the first trial (Figure S1). Means values for AUDPC in plants treated with chitosan oligosaccharide ranged from 118.92 to 276.91, with the highest value presented in the treatment chi -2 in the first trial and chi -5 in the second, and for tebuconazole in inoculated plants, mean AUDPC values ranged from 37.20 to 89.60 (Figure 2 A and B). In general, both fungicide and chitosan oligosaccharide performed best in the first trial but only fungicide significantly reduced AUDPC compared to the NTC. No FHB symptoms were presented in the mock-inoculated plants in both trials.\u003c/p\u003e\n\u003cp\u003eDON was detected in all samples of bulked kernels, except in the mock-inoculated control (\u003cem\u003edata not shown\u003c/em\u003e). There was an association between the reduction of mean AUDPC (Figure 1) and reduction in DON accumulation wheat grains (Figure 2 C and D). In general, some treatments with chitosan oligosaccharide suppressed DON accumulation compared to the NTC. For example, the treatments Chi -5 in the first trial and Chi 0 and Chi +2 in the second trial, with reduction ranged from from 13.6 to 42.3%. Additionally, fungicide treatments were able to suppress DON accumulation from 49 to up to 97.1%.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eField assay\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Overall, there was a large variation in the data which may have prevented us from detecting any significant difference between the treatments. Although not significant, there was a slight reduction in FHB severity on inoculated wheat heads treated with chitosan oligosaccharide or tebuconazole. The highest reductions in disease severity were observed in plants treated with fungicide (44.5 to 100%) regardless of when it was applied. For plants treated with chitosan oligosaccharide, highest reduction in FHB severity was observed in the plants sprayed two days before inoculation (49.70%) followed by treatments applied on the day of inoculation (39.30%). On average, all treatments were able to reduce in relation to the nontreated check, ranging from 12.0 to 100%, except for chitosan oligosaccharide applied five days before inoculation (Figure 3).\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eBiostimulant effect\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eThere was no significant effect of chitosan oligosaccharide or even fungicide sprays in increasing treated plants height (\u003cem\u003eP \u0026gt;\u003c/em\u003e 0.05), regardless of the concentration tested (Table S2) or timing of those sprays (Table S3). The percentage of \u003cem\u003eFusarium\u003c/em\u003e-damaged kernels ranged from 0 to 48.5% across all trials. Tebuconazole treatments significantly reduce FDK in inoculated plants across all trials (Table S4 and S5) compared to the NTC, except for the field trial (Table S6). Conversely, chitosan oligosaccharide regardless of the concentration tested (Table S5) or timing of those sprays (Table S4) did not significantly reduce FDK compared to the NTC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThousand-kernel weight ranged between 25.80 and 53.20% across all trials (Tables S7, S8 and S9). Mean TKW was highest in plants treated with tebuconazole sprayed in different timings (Table S3). For chitosan oligosaccharide, some concentrations and timings were able to prevent TKW reduction of up to 50% under greenhouse conditions. There was no significant reduction in TKW in inoculated plants either sprayed with chitosan oligosaccharide or tebuconazole in the field (Table S9).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, the potential sole use of the chitosan oligosaccharide in FHB management as well as its effect in suppressing DON accumulation in grains was investigated. In the literature, the use of chitosan and its derivatives is well reported for the control of pathogens and diseases. Several studies have investigated the potential use of chitosan in a wide range of plant pathogens, either the direct effect during fungal cultivation \u003cem\u003ein vitro\u003c/em\u003e, for example \u003cem\u003eSclerotium rolfsii\u003c/em\u003e in carrots (Ahmed et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e), \u003cem\u003eBotrytis cinerea\u003c/em\u003e in kiwi (Hua et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e), \u003cem\u003ePseudoperonospora cubensis\u003c/em\u003e in cucumber (Henr\u0026iacute;quez-D\u0026iacute;az et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e), \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e in rice (Lopez-Moya et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e), or to be used in post-harvest treatment of \u003cem\u003eColletotrichum gloeosporioides, Colletotrichum acutatum, Fusarium oxysporum\u003c/em\u003e and \u003cem\u003ePhytophthora\u003c/em\u003e sp. in strawberry ((Melo et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e), \u003cem\u003ePenicillium citrinum\u003c/em\u003e and \u003cem\u003ePenicillium mallochii\u003c/em\u003e in orange (Coutinho et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e), \u003cem\u003eLasiodiplodia theobromae\u003c/em\u003e and \u003cem\u003eColletotrichum gloeosporioides\u003c/em\u003e avocado (Obianom et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e), and others.\u003c/p\u003e\n\u003cp\u003eThe incidence of \u003cem\u003eF. graminearum\u003c/em\u003e in seeds treated with tebuconazole and chitosan oligosaccharide, disinfested and not disinfested, were consistently reduced. The effect of chitosan oligosaccharide on the reduction of \u003cem\u003eF graminearum\u003c/em\u003e incidence may be caused by a direct effect on the pathogen infecting seed surface. The direct effect of chitosan in pathogens infecting plant tissue surfaces have already been reported in other pathosystems, for example on gray mold and blue mold caused by \u003cem\u003eBotrytis cinerea\u003c/em\u003e and \u003cem\u003ePenicillium expansum\u003c/em\u003e in tomato fruit (Liu et al. \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e). More studies are needed to investigate the potential of oligosaccharide to reduce the incidence of already established infections, either by direct effect or inducing plant resistance.\u003c/p\u003e\n\u003cp\u003eIn addition to controlling the pathogen, the chitosan oligosaccharide did not interfere with seed germination and, even if not significant, the chitosan oligosaccharide showed a slight increase in seed germination. Some studies in the literature mention that compounds derived from chitosan present can increase seed germination in several crops. Zohara et al. (\u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e) tested the use of chitosan as a biostimulant to control infection of cucumber by \u003cem\u003ePhytophthora capsici\u003c/em\u003e and observed that in addition to controlling the pathogens, chitosan improve the germination rate and some traits such as shoot and root weight. Mazaro et al. (\u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e) evaluated the effect of treating sugar-beet and tomato seeds with chitosan to control of damping-off in substrate infested with \u003cem\u003eRhizoctonia\u003c/em\u003e sp., as a result, they reported that there was a reduction in the incidence of the pathogen, reducing the number of plants felled, which may be related to the increase in phenylalanine ammonia-lyase, since it acts in the formation of many plant defense mechanisms. Further studies can be conducted to better understand the effect of the seed treatment with chitosan oligosaccharide in the development of the wheat plants.\u003c/p\u003e\n\u003cp\u003eIn the present study, the effect of chitosan oligosaccharide directly on \u003cem\u003eF. graminearum\u003c/em\u003e was investigated. Although the estimated EC\u003csub\u003e50\u003c/sub\u003e values were high compared to the fungicides included as controls, the tested isolate was sensitive to chitosan oligosaccharide as concentration was increased. These findings show that chitosan oligosaccharide can directly reduce both mycelial growth and macroconidia germination of \u003cem\u003eF. graminearum\u003c/em\u003e. The direct effect of chitosan in some pathogens, interfering with mycelial growth, have already been reported (Berger et al. \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). Previous studies have already reported the effect of chitosan inhibiting \u003cem\u003eF. graminearum\u003c/em\u003e mycelial growth and conidia germination \u003cem\u003ein vitro\u003c/em\u003e (Kheiri et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e) and the effect of oligochitosan and chitosan hydrochloride, other chitosan derivatives, to inhibit mycelial growth for \u003cem\u003eF. graminearum\u003c/em\u003e (Xu et al. \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e; Deshaies et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e) showing that as the concentration increased, the growth of the pathogen was affected.\u003c/p\u003e\n\u003cp\u003eIncreasing the chitosan oligosaccharide doses led to a reduction in pH values and an increase in electrical conductivity values. Sporangia and zoospores of \u003cem\u003ePhytophthora ramorum\u003c/em\u003e were tested in response to different electrical conductivity values. The authors showed that in conditions where the values of electrical conductivity and pH were high there was a reduction in the viability of \u003cem\u003eP. ramorum\u003c/em\u003e zoospores (Kong et al. \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). Another study on the use of biochar showed that the use of this compound in the soil significantly increased the soil pH, electrical conductivity and other parameters. Furthermore, an increase in bacteria that promote biocontrol, such as \u003cem\u003eBacillus subtilis\u003c/em\u003e and suppression of pathogens of the genera \u003cem\u003eFusarium\u003c/em\u003e and \u003cem\u003eIlyonectria\u003c/em\u003e, was observed (Wang et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eIn the greenhouse experiments, chitosan oligosaccharide was sprayed in different timings around anthesis when the inoculation was performed. Kheiri et al.(2016) applied chitosan on wheat plants in 0, 3 and 5 days by spraying after inoculation. They observed that there was a reduction in severity of around 30% in treatments with chitosan about the control also failing to differentiate the influence of the application period on disease control. Deshaies et al. (\u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e) observed that even when chitosan was applied pre or post inoculation in wheat plants for \u003cem\u003eF. graminearum\u003c/em\u003e, the control was effective, reducing AUDPC. Unfortunately, our data did not provide enough evidence to confirm this hypothesis. Although not significantly different from the NTC, there was a reduction in FHB severity and AUDPC in the plants sprayed five days before inoculation in the first trial and on the day of the inoculation in the second trial. These inconsistencies between them may be explained, at least in part, by weather conditions during each trial (data not shown). The disease progress rate was slower in the nontreated check in the first trial compared with the second, when the conditions were more conducive for the development of the disease. Further studies are needed to understand the best time of application of chitosan oligosaccharide to control FHB in wheat.\u003c/p\u003e\n\u003cp\u003eOur results suggest that some treatments were effective for the reduction of deoxynivalenol. In the first trial, in the treatment Chi \u0026minus;\u0026thinsp;5 there was a reduction of 33.1% and in the second, in the treatment Chi 0 of 42.3% and +\u0026thinsp;2 of 13.6% in the treatments that were applied to the chitosan oligosaccharide. Silva \u003cem\u003eet al.\u003c/em\u003e (2015) used a chitosan film to reduce the aflatoxin produced by \u003cem\u003eAspergillus parasiticus\u003c/em\u003e in peanuts. The authors reported that the reduction of aflatoxin in the grains was greater than 80%. Zachetti et al. (\u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e) tested the use of chitosan as an alternative to control the growth and mycotoxin of \u003cem\u003eFusarium verticilioides, F. proliferatum\u003c/em\u003e (fumonisin) and \u003cem\u003eF. graminearum\u003c/em\u003e (deoxynivalenol) production in corn and wheat and showed that chitosan had an effect on the reduction of these mycotoxins, as demonstrated in this study.\u003c/p\u003e\n\u003cp\u003eChitosan oligosaccharide did not increase control efficacy with the increase of the concentration sprayed in wheat plants. The plants treated with different doses of chitosan oligosaccharide, applied five days before inoculation some doses were able to reduce the severity of \u003cem\u003eF. graminearum\u003c/em\u003e in relation to the control, in the different days of evaluation and consequently reduce the AUDPC, but there were no significant differences between them. Zachetti et al. (\u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e) conducted an \u003cem\u003ein vitro\u003c/em\u003e study using different doses of chitosan to inhibit the mycelial growth of different \u003cem\u003eFusarium\u003c/em\u003e species. In their results, for \u003cem\u003eFusarium graminearum\u003c/em\u003e, they observed that, regardless of the dose used, there was no interference in the growth of the isolate. In our study, as discussed above, in the \u003cem\u003ein vitro\u003c/em\u003e tests we were able to observe a decrease in mycelial growth and macroconidia germination with the increase of chitosan oligosaccharide concentrations, which were not observed \u003cem\u003ein planta\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eAdditionally, the application of chitosan oligosaccharide did not result in increased plant height in all assays. Cancio et al. (\u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e) included in their study that chitosan-treated bean seeds did not show significant differences in plant height when compared to the control. The weight of 1000 grains was also evaluated. The treatments with chitosan oligosaccharide applied 5 days before inoculation showed an increase of 11.8% in the final weight of the grains. In the second trial, the treatments applied on the day of inoculation and 2 days after inoculation showed an increase of 14.76 and 7.05%. He et al. (\u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e) using chitosan oligosaccharide for pre-harvest treatment of strawberry and concluded that the product brought an increase in viscosity, lignin, sugar, protein, total soluble solids and titratable acidity in the fruits.\u003c/p\u003e\n\u003cp\u003eIn conclusion, in the \u003cem\u003ein vitro\u003c/em\u003e test, chitosan oligosaccharide was able to reduce mycelial growth and conidia germination at higher doses of the product. In the greenhouse and field trials, chitosan oligosaccharide was able to reduce the FHB intensity, although it did not differ from the control. The amount of deoxynivalenol accumulated in wheat grains inoculated under greenhouse conditions was reduced in some greenhouse treatments. Our results suggest that chitosan oligosaccharide has some direct effect on \u003cem\u003eF. graminearum\u003c/em\u003e, but further studies are needed to better understand the in vivo efficacy of this product in combination with fungicides.\u003c/p\u003e\n\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003cbr\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: G.F.P. and F.J.M.; methodology: G.F.P., F.J.M. and E.M.D.; investigation: G.F.P., L.L.S.A., R.A.S., J.M.S and F.J.M.; statistical analysis: G.F.P., F.J.M. and E.M.D.; writing - original draft preparation: G.F.P. and F.J.M.; writing - review and editing: G.F.P., L.L.S.A., R.A.S., J.M.S., F.J.M. and E.M.D.; supervision: F.J.M. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and R codes used in the analysis are available as a research compendium in https://github.com/Gabrielgt40/Analysis-paper-COS01.git\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Programa de P\u0026oacute;s-gradua\u0026ccedil;\u0026atilde;o em Fitopatologia (UFV) and Capes/Proex (Programa de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior/Programa de Excel\u0026ecirc;ncia Acad\u0026ecirc;mica) for providing a graduate scholarship to G. F. Paiva. We thank the ALAS (Am\u0026eacute;rica Latina Agricultura Sustent\u0026aacute;vel) for the financial support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn behalf of all authors, the corresponding author states that there is no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was partially funded by Capes/Proex (Programa de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior/Programa de Excel\u0026ecirc;ncia Acad\u0026ecirc;mica) and ALAS (Am\u0026eacute;rica Latina Agricultura Sustent\u0026aacute;vel). Graduate scholarship of G. F. Paiva was provided by CAPES.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhmed MU, Bhuiyan MKA, Hossain MM, et al (2019) AND IMPROVEMENT THE CROP PRODUCTION. 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Crop Prot 73:100\u0026ndash;107. https://doi.org/10.1016/j.cropro.2015.02.025\u003c/li\u003e\n\u003cli\u003eWood AKM, King R, Urban M, et al (2020) Genome Sequence of Fusarium graminearum Strain CML3066, Isolated from a Wheat Spike in Southern Brazil. Microbiol Resour Announc 9:. https://doi.org/10.1128/MRA.00157-20\u003c/li\u003e\n\u003cli\u003eXu J, Zhao X, Han X, Du Y (2007) Antifungal activity of oligochitosan against Phytophthora capsici and other plant pathogenic fungi in vitro. Pestic Biochem Physiol 87:220\u0026ndash;228. https://doi.org/10.1016/j.pestbp.2006.07.013\u003c/li\u003e\n\u003cli\u003eYin H, Du Y, Dong Z (2016) Chitin Oligosaccharide and Chitosan Oligosaccharide: Two Similar but Different Plant Elicitors. Front Plant Sci 7:\u003c/li\u003e\n\u003cli\u003eYin H, Zhao X, Du Y (2010) Oligochitosan: A plant diseases vaccine\u0026mdash;A review. Carbohydr Polym 82:1\u0026ndash;8. https://doi.org/10.1016/j.carbpol.2010.03.066\u003c/li\u003e\n\u003cli\u003eZachetti V, Cendoya E, Nichea M, et al (2019) Preliminary Study on the Use of Chitosan as an Eco-Friendly Alternative to Control Fusarium Growth and Mycotoxin Production on Maize and Wheat. Pathogens 8:29. https://doi.org/10.3390/pathogens8010029\u003c/li\u003e\n\u003cli\u003eZohara F, Surovy MZ, Khatun A, et al (2019) Chitosan biostimulant controls infection of cucumber by Phytophthora capsici through suppression of asexual reproduction of the pathogen. Acta Agrobot 72:. https://doi.org/10.5586/aa.1763\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eEstimates for the mean values (data from two experiments combined) of \u003cem\u003eF. graminearum\u003c/em\u003e incidence in wheat seeds.\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"565\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 117px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatments\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 117px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSurface sterilization\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInoculation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTebuconazole\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eChitosan oligosaccharide\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCheck\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 117px;\"\u003e\n \u003cp\u003eDisinfested\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eInoculated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e7.5 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e18 Bb*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e13.5 ABa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 117px;\"\u003e\n \u003cp\u003eNon disinfested\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e6 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e10 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e31.5 Bb*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 117px;\"\u003e\n \u003cp\u003eDisinfested\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNot inoculated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e6.5 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e8.5 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e12 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 117px;\"\u003e\n \u003cp\u003eNon disinfested\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e7.5 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e15.5 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e13.5 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe means followed by the lowercase letters in the columns and not followed by an asterisk in the columns did not differ from each other at 5% of significance by Tukey test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eCombined data from the two experiments, estimating the mean values of \u003cem\u003eF. graminearum\u0026nbsp;\u003c/em\u003eincidence in the germination assay in wheat seeds.\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"803\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 295px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIncidence (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 277px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSeed germination (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSurface sterilized\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInoculation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTebuconazole\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eChitosan oligosaccharide\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCheck\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTebuconazole\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eChitosan oligosaccharide\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCheck\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eDisinfested\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eInoculated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.99 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e1.49 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e3.98 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e65 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e74 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e69 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eNon disinfested\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e6.33 Bb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e28.52 Cb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e77 Bb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e77 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e55 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eDisinfested\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNot inoculated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.46 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e3.26 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e0.30 Aa*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e80 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e72 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e80 Aa*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eNon disinfested\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.10 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e2.27 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e0.46 Aa*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e80 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e85 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e81 Aa*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe means followed by the lowercase letters in the columns and not followed by an asterisk in the columns did not differ from each other at 5% of significance by Tukey test.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"tropical-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tppa","sideBox":"Learn more about [Tropical Plant Pathology](https://www.springer.com/journal/40858)","snPcode":"40858","submissionUrl":"https://www.editorialmanager.com/tppa","title":"Tropical Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Wheat scab, Triticum aestivum, Alternative control, Integrated management of plant diseases","lastPublishedDoi":"10.21203/rs.3.rs-7915619/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7915619/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFusarium head blight (FHB), caused by members of the \u003cem\u003eFusarium graminearum\u003c/em\u003e species complex, is one of the most serious wheat diseases worldwide, capable of causing severe yield losses and affecting grain quality. Methods available for FHB management include cultural practices, sowing of resistant cultivars, chemical control, and biological control. In the present study, we evaluated the effect of chitosan oligosaccharide on the control of fungal growth, seed infection, disease symptoms and mycotoxin contamination under both controlled environments and in the field. In the in vitro conditions, chitosan oligosaccharide directly affected fungal growth at different levels depending on the concentration used. When applied as a seed treatment, both tebuconazole and chitosan oligosaccharide reduced the incidence of \u003cem\u003eF. graminearum\u003c/em\u003e and preserved seed germination potential of infected seeds. In the field, there was no significant difference in disease severity and DON in grains between the unsprayed check and the fungicide, regardless of the doses or times of chitosan oligosaccharide application. The results of this study suggest that chitosan oligosaccharide can potentially be used as a tool in the integrated management of the disease, establishing a basis for future studies to explore its use associated with chemical products, aiming to increase the efficacy of FHB control in wheat.\u003c/p\u003e","manuscriptTitle":"Evaluation of chitosan oligosaccharide as an alternative treatment to mitigate Fusarium graminearum and mycotoxin contamination in wheat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-25 09:27:38","doi":"10.21203/rs.3.rs-7915619/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-12-22T08:48:34+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-11-14T01:02:57+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-13T12:28:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Tropical Plant Pathology","date":"2025-11-13T09:06:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Tropical Plant Pathology","date":"2025-10-27T08:38:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"tropical-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tppa","sideBox":"Learn more about [Tropical Plant Pathology](https://www.springer.com/journal/40858)","snPcode":"40858","submissionUrl":"https://www.editorialmanager.com/tppa","title":"Tropical Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2539758d-4767-4ba2-95c6-eac117f0df41","owner":[],"postedDate":"November 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-19T12:26:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-25 09:27:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7915619","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7915619","identity":"rs-7915619","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}