**1. Introduction**

Grapevine trunk diseases reduce the lifespan of vineyards and increase the costs of producing wine grapes [1]. They are caused mainly by fungal pathogens, with the major pathogens including *Phaeomoniella chlamydosporum*, *Phaeoacremonium aleophilum*, *Botryosphaeria* spp., *Cylindrocarpon* spp., *Eutypa lata*, and *Phomopsis viticolaand* [2].

Esca is a destructive grapevine trunk disease first described over 100 years ago, which occurs worldwide and induces heavy economic losses [3]. The disease could be developed by intensive pruning, frost, and other mechanical injuries. The first symptoms of esca appear as dark red or yellow stripes on leaves, which eventually dry and become necrotic. The disease can then progress, potentially causing the entire plant to die [4]. Esca was first successfully controlled in 1903, when sodium arsenite was used as an insecticide on grapes. However, sodium arsenite was noted as being highly toxic and carcinogenic, and since 2003 has been banned in Europe. Nowadays, in practice, there is no curative approach for fighting esca. This fact challenges researchers to find a solution to effectively fight with this complex disease.

Electrospinning is a facile and efficient technique for fabricating functional nanofibrous materials possessing a large specific surface area and a fine porous structure [5,6]. These fibers have received much attention for use in many applications: Biomedical applications such as drug delivery [7], tissue engineering [8], and wound dressing, as well as cosmetics

**Citation:** Nachev, N.; Spasova, M.; Tsekova, P.; Manolova, N.; Rashkov, I.; Naydenov, M. Electrospun Polymer-Fungicide Nanocomposites for Grapevine Protection. *Polymers* **2021**, *13*, 3673. https://doi.org/ 10.3390/polym13213673

Academic Editor: Evgenia G. Korzhikova-Vlakh

Received: 30 September 2021 Accepted: 22 October 2021 Published: 25 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and functional materials and devices such as composite reinforcement, filters, protective clothing, and smart textiles, and even energy and electronics such as batteries/cells, capacitors, sensors, and catalysts.

Recently, novel varieties of the electrospinning technique were developed in order to generate more complex nanostructures with desired features such as coaxial electrospinning [9], side-by-side [10] and tri-axial [11] electrospinning, and other multiple fluid processes. Such methods of fabrication can lead to composite structures such as core– shell, Janus, tri-layer core–shell, and other complex structures. Another recently used strategy is to combine electrospinning with other traditional methods to fabricate novel nanofibers [12]. Regardless of the direction, the final objective is a suitable application of the resultant nanofibers [13]. The present study highlights a new potential application of electrospun nanofibers for grapevine protection.

Phytopathogenic fungal infections have become a serious problem in agricultural production, reducing food yield and quality. Therefore novel antifungal agents with high efficiency and low toxicity are needed. Modern plant protection products should be designed to achieve the desired biological effect without harmful impact or side effects. The use of nanomaterials in agriculture and, in particular, for the protection of vineyards is an emerging field of interest. Sett et al. created rayon membranes on which nanofibers of soy protein/polyvinyl alcohol and soy protein/polycaprolactone are electrospun. This material aims to physically block the penetration of fungal spores [14]. However, the authors commented that the blocking was insufficient and an antifungal component should be incorporated. Furthermore, electrospun materials from two copolymers loaded with polyhexamethylene guanidine have been fabricated to serve as bandages for vineyards against the penetration of esca-causing fungi [15]. The authors reported, however, that more effective polymers and antifungal agents should be used.

Some authors have also proposed an original approach and have successfully obtained fibrous materials from poly(3-hydroxybutyrate), nanosized TiO2-anatase, and chitosan oligomers (COS) with antifungal activity for plant protection via a combination of electrospinning and electrospraying [16]. The obtained eco-friendly materials possess high roughness, hydrophobicity, and antifungal activity against *P. chlamydospora*.

It is known that compounds containing 8-hydroxyquinoline exhibit anticancer [17,18], antimicrobial [19,20], antiviral [21], and antifungal activities [22,23]. Due to their antifungal properties, these biologically active compounds have found application in agriculture. We created stable solutions on the basis of a water-soluble polymer and a fungicide with antifungal activity suitable for applications in agriculture [24].

The incorporation of low molecular weight derivatives of 8-hydroxyquinoline into fibrous polymer materials obtained by electrospinning is of interest because it allows combining the valuable biological properties of 8-hydroxyquinoline derivatives with the advantages of electrospun materials. In our previous study, we obtained fibrous membranes on the basis on cellulose acetate loaded with an antifungal agent for active protection against spore penetration and plant infection in vineyards [25]. However, the incorporation of low molecular weight derivatives significantly lower the physicochemical properties and therefore they need to be improved.

In the last few decades, considerable research interest has been dedicated to the use of biodegradable polymer materials for various applications, such as in medicine, as well as in industry to replace conventional petrochemical-based polymers. Due to its merits, thermoplastic aliphatic polyesters are the most commonly explored synthetic biodegradable polymers. PLA has an extensive mechanical property profile and is a highly biocompatible and biodegradable polymer [26].

Therefore, the present study aimed to prepare electrospun composite materials from PLA and 8-hydroxyquinoline derivatives with antifungal activities. The effect of the incorporated biologically active compound on the morphology, wetting, crystallinity, thermal, and physicochemical properties was studied. Microbiological tests against *P. chlamydospora* and *P. aleophilum* were performed.

#### **2. Materials and Methods**

#### *2.1. Materials*

Poly(L-lactide) (PLA; Ingeo™ Biopolymer 4032D, NatureWorks LLC—USA, Minnetonka, MN, USA; M<sup>W</sup> = 259,000 g/mol; MW/M<sup>n</sup> = 1.94; as determined by size-exclusion chromatography using polystyrene standards), 5-nitro-8-hydroxyquinoline (Pharmachim, Sofia, Bulgaria), and 5-chloro-8-hydroxyquinoline (Sigma-Aldrich, St. Louis, MO, USA) were used. Dichloromethane (DCM; Merck, Darmstadt, Germany) and ethanol (abs. EtOH; Merck, Darmstadt, Germany) were of an analytical grade of purity.

Potato dextrose agar medium was obtained from Merck, Darmstadt, Germany. The disposable consumables were purchased from Orange Scientific, Braine-l'Alleud, Belgium.

### *2.2. Procedures*

The potassium 5-nitro-8-hydroxyquinoline was prepared as described by Ermakov and coworkers [27].

#### *2.3. Preparation of Electrospun Fibrous Materials*

Spinning solutions in DCM/EtOH (DCM/EtOH = 90/10) were prepared for PLA, PLA/5-Cl8Q, and PLA/K5N8Q. The total polyester concentration was 10 wt% 5-Cl8Q and 10 wt% K5N8Q.

A Brookfield LVT viscometer equipped with an adaptor for small samples, a spindle, and a camera SC 4-18/13 R at 20 ± 0.1 ◦C was used to measure the solution viscosities. The spinning solutions were measured in triplicate and the mean values with their standard deviations were used.

Electrospinning was performed using a high-voltage power supply (up to 30 kV), a grounded metal drum collector, an infusion pump (NE-300 Just InfusionTM Syringe Pump, New Era Pump Systems Inc., Farmingdale, NY, USA) for delivering the spinning solution at a constant rate, and a syringe equipped with a metal needle (gauge: 20GX1<sup>1</sup> 2 "). The applied voltage was 25 kV, the distance to the collector was 15 cm, the collector rotating speed was 1000 rpm, the humidity was 50%, and the temperature was 20 ◦C.

#### *2.4. Characterization of the Electrospun Materials*

The morphology of the fibers was examined by a scanning electron microscope (SEM). The samples were vacuum-coated with gold and observed by a Jeol JSM-5510 SEM (Jeol Ltd., Tokyo, Japan) at acceleration voltage of 10 kV with 1000×, 2500×, and 5000× magnification. The fiber morphology was evaluated using the criteria for complex evaluation of electrospun materials as described elsewhere [28] via ImageJ software by measuring the diameters of at least 20 fibers from each SEM micrograph [29].

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra were recorded using an IRAffinity-1 spectrophotometer (Shimadzu Co., Kyoto, Japan) equipped with a MIRacle™ATR (diamond crystal with a depth of penetration of the IR beam into the sample of approximately 2 µm) accessory (PIKE Technologies, Fitchburg, WI, USA) in the range of 600–4000 cm−<sup>1</sup> and a resolution of 4 cm−<sup>1</sup> . All spectra were corrected for H2O and CO<sup>2</sup> using an IRsolution software program.

The absence/presence of a crystalline phase in the electrospun materials was assessed by X-ray diffraction analysis (XRD). XRD spectra were recorded at r.t. using a computercontrolled D8 Bruker Advance powder diffractometer (Bruker, Billerica, MA, USA) with a filtered CuKα radiation source and a luminescent detector. The analyses were performed in the 2θ range from 5◦ to 50◦ with a step of 0.02◦ and a counting time of 1 s/step.

Static contact angle measurements of the membranes were performed using an Easy Drop DSA20E Kr˝uss GmbH drop shape analysis system (Hamburg, Germany) at 20 ± 0.2 ◦C. A sessile drop of deionized water with a volume of 10 µL controlled by a computer dosing system was deposited onto the electrospun fibrous materials. The contact angles were calculated by computer analysis of the acquired images of the droplet. The data are an average from 10 measurements for each sample.

Mechanical properties were evaluated by tensile measurements performed on the fibrous materials using a single-column system for mechanical testing INSTRON 3344, equipped with a loading cell 50 N and Bluehill universal software (Instron Bluehill Universal V4.05 (2017) software, Norwood, MA, USA). The stretching rate was 10 mm/min, the initial length between the clamps was 40 mm, and the room temperature was 21 ◦C. All samples were cut into dimensions of 20 <sup>×</sup> 60 mm<sup>2</sup> with a thickness of ca. 200 <sup>µ</sup>m. For the sake of statistical significance, 10 specimens of each sample were tested, after which the average values of Young's modulus, the ultimate stress, and the maximum deformation at break were determined.

5-Cl8Q and K5N8Q release was studied in vitro at 37 ◦C in acetate buffer (CH3COONa/ CH3COOH) containing lactic acid (acetate buffer/lactic acid = 96/4 *v*/*v*) at pH 3 and an ionic strength of 0.1. Fibrous materials loaded with 5-Cl8Q or K5N8Q (4 mg) were immersed in 100 mL of buffer solution under stirring in a water bath (Julabo, Seelbach, Germany). The release kinetics were determined by withdrawing aliquots (2 mL) from the solution at determined time intervals, then adding back the same amount of fresh buffer and recording the absorbance of the aliquots by a DU 800 UV–vis spectrophotometer (Beckman Coulter, Brea, USA) at wavelengths of 255 nm and 364 nm. The amount of released 5-Cl8Q or K5N8Q was calculated using calibration curves (correlation coefficient *R* = 0.999) for the membranes in acetate buffer/lactic acid = 96/4 *v*/*v* with a pH of 3 and an ionic strength of 0.1. The data are the average values from three measurements.

#### *2.5. In Vitro Antifungal Assay*

The antifungal activity of the fibrous materials was monitored against the fungi *P. chlamydospora* CBS 239.74 and *P. aleophilum* CBS 631.94. *P. chlamydospora* CBS 239.74 and *P. aleophilum* CBS 631.94 were purchased from Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands.

*P. chlamydospora* and *P. aleophilum* grow normally on potato dextrose agar [30] and malt extract agar [31].

In the present study, in vitro studies were performed using potato dextrose agar medium (PDA; Merck, Germany). The surface of the solid agar was inoculated with a suspension of fungi culture with a fungi concentration of 1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL, and on the surface of the agar in each Petri dish, one electrospun material (17 mm in diameter) was placed. The Petri dishes were incubated for 96 h for *P. chlamydospora* and *P. aleophilum* at 28 ◦C, and subsequently, the zones of inhibition around the disks were measured. The average diameters of the zones of inhibition were determined using ImageJ software based on 15 measurements in 15 different directions for each zone.

For the preparation of the conidia suspension test, microorganisms were grown on potato dextrose agar (PDA) medium for 14 days. Conidia were obtained by pouring 5 mL of sterile water onto the plate and washing it off with a sterile loop. Conidia suspensions were filtered through two layers of sterile round cloth to remove mycelial fragments. The final concentration of conidia was adjusted to 10<sup>7</sup> conidia/mL with sterile water. The fibrous materials were cut into disks with a diameter of 45 mm and a thickness of ~1 µm. Digital Thickness Gauge FD 50 (Käfer GmbH, Villingen-Schwenningen, Germany) was used to determine the thickness of the fibrous materials. All fibrous materials were sterilized for 30 min under UV light in a laminar box before being used for further experiments. Then, the fibrous material in disk form was placed between the two parts of the filtration device supplied with a pump. The two parts of the device were pinched with a clip. After this, 20 mL of the spore conidia suspension was passed through each type of fibrous material. Then, every used disk was taken with pincers and placed on a surface of solid PDA medium in a Petri dish. The Petri dishes were maintained at 28 ◦C for 96 h. Then, the fungal growth was assessed. The concentration of conidia passed through the materials was determined using a hemocytometer.
