Evaluation of antifungal activity of chitosan nanoparticles against Fusarium solani phytopathogenic fungi in in vitro and in vivo assays

Key message : We report that chitosan-TPP-PEG nanoparticles generate alterations at structural and functional level of the wall cell of Fusarium solani in in vitro and in vivo assays through TEM. Abstract Fungal diseases are a current problem in agriculture causing significant losses in several crops whereby its prevention and treatment is of utmost importance. The Chitosan nanoparticles (ChNPs) were evaluated for their antimicrobial activity against the phytopathogen Fusarium solani . The chitosan concentration in nanoparticles that showed antifungal activity was 2.0 µg/mL. ChNPs showed to be a potential antifungal candidate with applications in phytosanitary control. Transmission electron microscopy (TEM) results showed damage to the fungal cell wall and membrane caused by the nanoparticles interaction with these structures affecting fungal growth and development in i n vitro as in in vivo assay where microscopy demonstrated the internalization of nanoparticles aggregates within plant root cells cytoplasm up to 45 days. Therefore ChNPs nanoparticles could be an alternative method for diseases caused by Fusarium solani instead of chemical fungicides commonly used for treating tomato root rot.


Introduction 1
Incidence of pests and diseases is one of the most important troubles in world agriculture, as they cause significant losses in crops and threatening food security . Colombia is a mainly agricultural country favoured by its climatic conditions, where is common for vegetable farming being affected by a high number of agricultural pests and diseases (ICA, 2015).
Phytopathogenic fungi are responsible for the diseases with the highest incidence in different crops in the agricultural sector. These diseases are mainly caused by the inadequate management of the soils and water sources used for agriculture, in addition to the widespread use of pesticides which generate resistance in the different pathogens by adaptation to the chemical agent (Barrientos, 2009) becoming generalized diseases that affect agricultural production and consequently generating economic losses.
Fusarium spp. are phytopathogenic fungi that can be dispersed by different routes, contaminated seeds, wind, manipulation of the crop, diseased plants or the use of work elements that are contaminated. Fusarium spp. can survive in the soil for years (López, 2016, p. 69), this condition allows the infection of plants through the roots and can affect various types of crops such as vegetables, tubers and legumes. In Colombia, Fusarium spp. can cause losses between 21% and 47% in some monocultures (Vásquez & Castaño, 2017).
Among these phytopathogenic fungi, Fusarium solani, which has a high agricultural and medical relevance, also affects different crops such as peas (Adriana & Jairo, 2011), beans (Guerrero & Guerra, 2010) and tomato, in this case it has been reported as one of the main causes of root rot of the tomato in plants resistant to Fusarium oxysporum (Montealegre, Donoso, Herrera and Besoain, 2003).
Currently, Good Agricultural Practices (GAP) and pest and disease control techniques are being implemented as measures to prevent their spread. However, chemical control agents such as chlorothalonil, carbendazim, mancozeb and thiabendazole are also used (Elosan et al. , 2007); its irrational use is generating serious environmental consequences. Therefore, it is important to develop effective and sustainable alternative biological control treatments against this type of pathogens that involve the use of biopesticides in farming systems.
In recent years, the implementation of nanotechnological systems has emerged as an innovation in the field of biopesticides, some of them are based on chitosan nanoparticles (ChNPs). Studies report that chitosan is a compound with high antifungal activity (Carolyn R. Allan 1979;Pavel et al., 1999;Cotae and Creanga, 2005), it can affect the fungus through various forms of interaction. Chitosan interacts with the chitin wall of the fungus such as cell wall and membrane, generating metabolic stress and consequently affecting its development, formation and structure.
In this work, we evaluate the antifungal activity of ChNPs nanoparticles against the F. solani fungus under in vitro and in vivo conditions. The growth inhibition of the mycelium of F. solani treated with ChNPs and in comparison with Mertect a commercial fungicide was evaluated. The cellular ultrastructure of F. solani and its interaction with the ChNPs nanoparticles were analyzed by TEM. The antifungal activity of ChNPs was also evaluated in in vivo conditions with the Solanum lycopersicum plant affected by F. solani. TEM images were obtained showing the interaction of ChNPs agglomerates with the root of the plant.

Chitosan nanoparticles
Chitosan nanoparticles (ChNPs) were prepared by a research group of nanotechnology at the Universidad El Bosque, using 2.0mg/ml of low molecular chitosan, Acetic Acid at 0.15%v/v, sodium tripolyphosphate (TPP) as a crosslinking agent at 1 0.0 mg/ml and PEG at 50 mg/ml.

Fungal material preparation
Fusarium solani strain identified as Fusarium keratoplasticum Geiser et al. (ATCC 36031) belonging to the species bank was provided by the Universidad el Bosque. The strain was reactivated 7 days in advance on PDA medium at 25 ° C.

Determination of antifungal activity of ChNPs against F. solani in vitro
The inhibitory effect of 2.0mg/ml chitosan in nanoparticles (ChNPs) on radial growth and germination of conidia of the fungus F. solani was evaluated in in vitro tests. Mertect ® 500 sc (Sygenta S.A) was used as a commercial fungicide.

Effect on mycelial growth
For the evaluation of the effect of ChNPs on mycelial growth, the agar dilution method was implemented as proposed by Saharan, et al. (2015). 100μl of the treatment (Pardo, 2017), ChNPs in a 1/100 ratio or Mertect ® 500 sc (0.5mg/ml), were added to the Petri dishes with PDA medium in liquid phase then it was distributed evenly and allowed to solidify. Subsequently, agar mycelial discs were taken from a 7 days old culture, placed in the middle of the petri dish and incubated at 25 ° C for 12 days. This procedure was performed in triplicate for each treatment. Mycelial growth data was recorded during the evaluation time and %inhibition was calculated using the formula (Juniors et al., 2017) (Sathiyabama, 2016).

Effect on conidia germination
A conidial count was made for one of the replicas of each treatment from the above procedure. 0.85% NaCl was added to the culture medium, then conidia were carefully scraped off from the medium surface and condial counting with Neubauer chamber was performed (Saharan, et al., 2015).

Plant material growth conditions and antifungal evaluation of ChNPs
Plant material used were chonto tomato seedlings obtained at the Jorge Tadeo Lozano University Biosystems Center with approximately 15 to 20 days of age.

Fungus inoculation
A suspension of 1x10 5 conidia/ml of F. solani was prepared for inoculated the tomato plants by Root Dipping (RD), (El-mohamedy et al., 2014) (López Benítez et al., 2016 submerging the roots for 2 min, then they were transplanted into containers with sterile soil (López Benítez et al., 2016). This was made for triplicate for each of the treatment (Healthy, infected plant and treatment with ChNPs).

Application of ChNPs
After two weeks of plant infestation, ChNPs were applied by using root dipping. In the case of root dipping, tomato plants roots were dipped for 30 minutes in a ChNPs suspension. Then the seedlings were transplanted again to soil infested with F. solani (Elmohamedy, Abdel-Kareem, Jaboun-Khiareddine, & Daami -Remadi, 2014).) These techniques were carried out for triplicate and evaluated after a period of 45 days. In the first 12 th h of the application a root sample was taken so it can be observed the internalization of ChNPs in an early stage

Transmission Electron Microscopy Method
TEM was used to examine the size and morphology of the ChNPs nanoparticles used in each treatment. TEM was also used to evaluate the ultrastructure of F. solani cells treated with ChNPs, with Mertect and untreated cells.
For the preparation of the samples obtained for visualization at the ultrastructural level of F. solani, an inoculum of 1x10 6 conidia/mL in NaCl at 0.85% w/v was cultured in YPG liquid medium at 25 °C for 6 h, then 2mL ChNPs nanoparticles were added and they were incubated at 25 °C for 12 h with constant shaking. Then the cells were centrifuged at 3000 rpm for 10 min, washed twice with PBS (phosphate buffered saline) 0.1M at pH 7.5 and finally fixed with glutaraldehyde at 2.5% v/v, as reported by Zhang et al. (2019) and Gong et al., (2015). Positive controls were performed with the fungicide Mertect at 0.5 mL/ml (concentration currently used in agriculture) and negative controls with YPG plates without the presence of ChNPs. All cultures were carried out under the same conditions and each procedure in triplicate.
Likewise, the ultrastructure of the plant root cells infected with F. solani and treated with ChNPs was evaluated by TEM, as controls infected cells not treated with ChNPs and non-infected cells were evaluated. The samples were prepared as reported by YP Zhang et al., (2018), 2mm 3 sections of the plant root with signs of rot were cut and fixed by immersion in glutaraldehyde at 2.5% v/v. The following process of preparing the samples for TEM was carried out by the company that developed the microscopy Fundación Santa Fe de Bogotá (Colombia), each sample was fixed in 2% osmium tetroxide (OsO4) for 1 h, then dehydrated by washing with serial concentrations of ethanol and finally, inclusion in plastic with a semi-fine section was made for consequent visualization in the Jeol JEM-1400 plus TEM microscope.

Statistical analysis
Percentage of inhibition data obtained were statistically analyzed using the ANOVA One Way test using SPSS. A Tukey a test was carried out with p <0.5 to determine statistical differences between the treatments.

Examination of the ChNPs nanoparticles
ChNPs were characterized by transmission electron microscopy TEM, agglomerates of ChNPs nanoparticles with sizes of 50-100 nm are shown ( Fig. 1).

Antifungal activity of ChNPs against F. solani
We show in this work the antifungal activity of chitosan nanoparticles conjugated with TPP and PEG synthesized by a research group from El Bosque University (Colombia). We evaluated the antifungal activity of ChNPs against the fungus Fusarium solani under in vitro conditions. Samples with F. solani treated with ChNPs were analyzed and as controls F. solani cells without treatment and F. solani cells treated with the commercial fungicide Mertect.

Effect of ChNPs nanoparticles on the mycelial growth
The control group shows in vitro normal growth of F. solani (Fig. 2a) when compared with the group treated with ChNPs, inhibition of the growth of F. solani is observed, the reduction in colony size is significant (Fig. 2b), and an inhibitory effect is also shown in the group supplemented with Mertect, a drug against F. solani (Fig. 2c).
The percentage of inhibition of F. solani was calculated in each treatment. The nanoparticles showed antifungal activity significantly inhibiting the radial growth of F. solani at 240h of exposure, where an average value of 7.40% was presented compared to Mertect, which presented 14.11% (p <0.05). Results Tukey significance level QNP2 with respect to the mertec (72h) 0,727 (240h) 0.785 (Fig. 3).

Effect of ChNPs nanoparticles on conidia germination
The addition of ChNPs to the PDA culture significantly affected the germination of F. solani conidia in vitro culture. The quantification of conidia in the treatment with nanoparticles was 9.6x10 5 conidia/mL compared to 3.1x10 6 with Mertect and 7.4x10 6 in the control group. The results indicated that the addition of 100µL of nanoparticles to the culture medium was effective for inhibit the germination of conidia in the tests more than Mertect, which is a drug currently used as fungicide (Fig. 4).

Effects of ChNPs on the ultrastructure of F. Solani
TEM was used to evaluate the effect of ChNPs on the cell structure of the F. solani fungus in vitro. F. solani cells were examined without treatment and supplemented with ChNPs (2mL). In figure 5 (a-c) untreated fungal cells are observed, complete integrity of the cellular structure is shown, in which the cell wall and the plasma membrane are clearly defined. The cytoplasmic content shows a normal density as reported by Benhamou & Be (1998), evidencing its cellular structures, mainly the nucleus, vacuoles and lipids. Also was possible to detect septa of the fungal hyphae.
The treatment of F. solani with ChNPs nanoparticles showed the presence of agglomerates of nanoparticles surrounding F. solani cells (Fig. 5d,g,h). This polymeric material generally established close contact with the cell wall and the plasma membrane of the fungus where its structure is strongly altered. As a consequence of this interaction, the disintegration of these two cellular structures is generated each losing its integrity (Fig. 5e, g, h, red arrow) compared to the control sample in which a defined structure of both the plasma membrane and the cell wall is presented (Fig. 5c, arrow). Another modification generated by the effect of nanoparticles is the thickening of the cell wall (Fig. 5f, red arrow).
Treatment with nanoparticles lasting 12 hours showed that F. solani cells do not present significant changes in some of their cellular structures, the Golgi apparatus, and endoplasmic reticulum have normal morphology (Fig. 5g, h respectively), however, it was observed that the nucleus increased its size in cells treated with nanoparticles ( Fig. 5e) compared to untreated cells (Fig. 5c). F. solani cells were also evaluated with Mertect fungicide treatment to compare the antifungal activity of the ChNPs nanoparticles.
In Figure 6 (a-e) fungal cells treated with Mertect with morphological alteration were observed. Some cells showed increase in the amount of lipid in the cytoplasm (Fig. 6a) compared to untreated cells (Fig. 5b). Some cells treated with Mertect also showed loss of part of the plasma membrane and cell wall (Fig. 6b), other cells showed disruption in part of their plasma membrane and in the cell wall (Fig. 6d). Similar results were observed in the treatment with ChNPs where the interaction with the ChNPs strongly affected the integrity of the plasma membrane and cell wall (Fig. 6f-i red arrow). Likewise, fungal cells treated with Mertect presented loss of consistency of the cytoplasm (Fig. 6c (Cy) and some cells presented high-density granules inside this (Fig. 6d). The same way the cytoplasmic content of the fungal cell treated with nanoparticles was affected, high-density granules were also observed within the cytoplasm of some cells (Fig. 6f, h). Another cellular structure of F. solani strongly affected each treatment was the nucleus, which considerably increased its size both with the presence of ChNPs nanoparticles (Fig. 5e) and Mertect (Fig. 6b, e).  (Fig. 6 a, b, e). Loss of the plasma membrane, cell wall (Fig. 6b) and consistency of the cytoplasm (Fig. 6c (Cy) occurs in some cells. Disruption of plasma membrane and cell wall integrity was visible in some cells (Fig. 6d). Increase in amount of lipids (Fig. 6a). (f-i) cell with ChNPs nanoparticles 2ml. The presence of agglomerates of nanoparticles generated a strong alteration in the integrity of the plasma membrane and cell wall of the fungal cells (Fig. 6f, g, i). Distribution of the agglomerates of nanoparticles surrounding the cell wall interacting and affecting its integrity (Fig. 6g, h,

Effects of ChNPs nanoparticles on the physiology of Solanum lycopersicum
Slight signs of tomato root rot were observed in Figure 7a where the root treated with the ChNPs has a larger number of root hairs compared to the plant root that was not treated with the nanoparticles (Figure 7b) as well as the presence of secondary roots and therefore a biomass increase in contrast to the plant without the treatment, where it was evidenced the necrosis of the foot, lack of secondary roots and a decrease in the amount of root hairs.

Effects of ChNPs nanoparticles on the ultrastructure of root of Solanum lycopersicum
TEM microscopy was also used to assess the effect of ChNPs nanoparticles on the root of the Solanum lycopersicum plant infected by the fungus F. solani. Initially, the cell structure of the plant root was compared in healthy conditions (without infection) and in the presence of the fungus, images were captured that showing the changes generated in the cellular structure. Figure 8a y 8b shows cell structures of the plant root in healthy conditions, the cell wall is clearly defined.
Figures 8c-f shown plant root infected by the fungus but not treated with ChNPs nanoparticles clearly cell structures of F. solani with normal appearance are observed, among them the cell wall, plasma membrane and the cytoplasm. These images are comparable with those obtained in the TEM of the in vitro samples of F. solani (Fig. 5a, c), in which the cell wall and the plasma membrane of some cells of F. solani were also observed.  Figure 8g shows morphologically altered plant cell structures. Fig. 8i shows various agglomerates of nanoparticles with sizes less than 200nm. Fig 8h, j, k shows Solanum lycopersicum root cells infected with F. solani, the presence of ChNPs nanoparticles are also shown inside the plant cell. Some nanoparticles are present inside the fungal cells that are infecting the plant (Fig. 8k), alteration of the cell wall and plasma membrane of the fungus generated by stress is observed as a consequence of the interaction with the nanoparticles (Fig. 8h, k). Figure 8k also showed high-density granules in cells colonized by F. solani, indicating that the cytoplasmic content of the plant cell is strongly affected by the presence of the fungus.
To compare the effect of ChNPs nanoparticles on F. solani in vivo for 12 hours post infection, Solanum lycopersicum root was exposed to nanoparticles during this time period. In Figures 9a and 9b the cell wall and plasma membrane of the plant cell are shown altered, likewise the cytoplasm of the plant cell is disrupted (Fig 9b). Figure 9c shows the plant cell infected with F. solani, the presence of ChNPs nanoparticles are also shown in cytoplasm the plant cell. Figure 9d shows F. solani cell structures near to the plant cell wall, loss of integrity of the cell wall and presence of extracellular vesicles, possibly as a consequence of the stress generated by the nanoparticles.  (Fig. h, j, k). Fig. i shows various agglomerates of nanoparticles with sizes less than 200nm. Some nanoparticles are presents inside the fungal cells that are infecting the plant (Fig. 8 k). . We show in this work the antifungal activity of chitosan nanoparticles conjugated with TPP and PEG synthesized by a laboratory. Ionotropic crosslinking with TPP defines the size and shape of the nanoparticle which allows adjusting these variables to synthesize chitosan nanoparticles with optimal antimicrobial activity (Wazed Ali S et al 2010).
In this work, we evaluated the antifungal activity of ChNPs against the fungus Fusarium solani under in vitro and in vivo conditions. Figure 1 shows agglomerates of nanoparticles with size between 50-200 nm, results comparable to those obtained by Kaloti & Bohidar (2010), Karimirad, Behnamian, & Dezhsetan (2019) and Omidi & Kakanejadifard (2019) where ChNPs agglomerates have been described with sizes between 30nm to 300nm. Similar results were also obtained by Ing, Zin, Sarwar and Katas (2012) reporting ChNPs with sizes between 150 and 250 nm with high antifungal activity.
The efficacy in using ChNPs as a fungal control method against F. solani growth was determined by in vitro evaluation. The ChNPs presented antifungal activity by inhibiting the growth of the fungus (Fig. 2b) compared to the control (Fig. 2a), similar results were obtained with the treatment with the commercial fungicide Mertect (Fig. 2c) However, the commercial fungicide had a greater inhibitory effect on the radial growth of the mycelium of the fungus with PIRG of 14.11% compared to the nanoparticles which was 7.40% (Fig 3). This may be due to the composition of the nanoparticles, since TPP and PEG interact with the amino groups of chitosan to form the nanoparticulate system that causes a slight decrease in its antifungal activity (N. Rodríguez, Valderrama, Alarcón, & López, 2010), considering that these free amino groups are responsible for generating antifungal effect (Cárdenas et al., 2015). The antimicrobial activity of chitosan depends on its molecular weight and degree of deacetylation, to determine the maximum antimicrobial activity of chitosan it is necessary to analyze a great variety of molecular masses of chitosan with the same degree of deacetylation (Riad SR El-Mohamedy et al 2019).
Nevertheless, the treatment with ChNPs significantly affected the conidia germination of F. solani, presenting approximately 87% reduction in conidia germination compared to the control without nanoparticles, while the fungicide Mertect presented a 58% reduction (Fig. 4). TEM allowed to observe the ultrastructure of F. solani (Fig 5a-c) is similar to those presented by Benhamou & Be, 1998;Maciá-Vicente, Jansson, Mendgen, & Lopez-Llorca, 2008;Pârvu, Barbu-Tudoran, Roşca-Casian, Vlase, & Tripon, 2010, where defined cell wall and membrane, as well as nucleus, presence of vacuoles and other organelles were observed in the cytoplasm, without any morphological alterations or irregularities. In contrast to those cells exposed to Mertect (Fig 6a-e) ultrastructural changes were observed in the wall and membrane of the fungus as well as high density granules at the cytoplasm, density of the nucleus and cytoplasm were affected due to the Thiabendazole. Also, larger vacuoles and an increase in the number of lipid vesicles were evidenced which could be a reaction due to the stress caused by thiabendazole to the fungal cell. Thiabendazole is the main compound of Mertect, the study of Andrioli NB & Mudry MD, 2011 reported that thiabendazole (TBZ) caused mitotic toxicity, their results showed that meristematic cells of Allium cepa exposed to TBZ presented toxicity and genotoxicity interfering with microtubule formation.
Translated with www.DeepL.com/Translator (free version) Nanoparticles are a colloidal system that effectively interacts with the fungal cell wall and membrane as showed in figures 5f-h and 6f-i. Chitosan nanoparticles are considered to have the antifungal activity from chitosan; this property is associated with the free amino groups interacting with the negatively charge macromolecules that compounds the cell wall from F. solani which affects its permeability as well as the cell membrane permeability, consequently causing the malfunction of the main protective barriers of the fungal cell (Cardenas et al., 2015) and inhibiting the mycelial growth (Fig 2). This damage to the cell wall can be watched at Figures 5, 6 and 8 where the nanoparticles agglomerates interacts with fungal wall and membrane causing disintegration of these structures as observed in TEM figures by losing their defined shape as seen in the figure 5 (a-c) where is untreated F. solani cells.
This effect of the nanoparticles on the fungal cell can be observed the in vitro model as inside the plant (Fig. 10), where the damage in the fungal cell wall can be presented not also when F. solani is exposed directly to the nanoparticles (Figure 10a) but also when this fungus is inside tomato roots infecting the plant (Figure 10b). The interaction that is evidenced between the vegetable tissue of the tomato plant and the chitosan nanoparticles favors the plant because this material is not only reported as an antimicrobial agent, but also as an elicitor to induce the systemic resistance induced in plants (Sharp, 2013;Katiyar at al, 2015;Benhamou and Thériault, 1992;Benhamou et al, 1994), protecting them from the production of defense responses and preparing the plant for next attacks of pathogens, within the defense mechanisms induced by chitosan particles are the production of reactive oxygen species such as hydrogen peroxide, nitric oxide, among others, this occurs due to the activation of phosphorylation and dephosphorylation chains of MAPK, which send signals to the nucleus thus activating the transcription factors (Lin et al, 2005;Li et al, 2009;Zhang et al 2011), another advantage of this direct interaction, is the induction of plant growth and increase in biomass yield (Wang et al, 2015) as showed in Fig 7a where an increase in secondary roots and root hairs was observed.
It is important to consider the implications of the use of nanotechnology in agriculture to avoid phytotoxicity as a consequence of exposing plants to nanoparticles treatments, in addition, specific tests should be considered to evaluate the effect of ChNPs entering the food chain. Actually, several studies have been reported that show the use of ChNPs nanoparticles for various applications in plants, some studies report the antifungal activity of ChNPs in plants for human consumption.
However, no studies of the use of ChNPs in Solanum lycopersicum are reported, in this work we show the internalization of these ChNPs in Solanum lycopersicum and they interaction with a phytopatogenic agent as it is Fusarium solani, exploring the use of these kind of ChNPs nanoparticles reticulated with TPP an PEG a method to treat diseases caused by this fungus and like an alternative to chemical pesticides.