43 Adv. Hort. Sci., 2022 36(1): 43­52 DOI: 10.36253/ahsc­10449 Phytoprotective film for resistance induction, growth, and yield of organic strawberries I. Dos Santos Pereira (*), F. Grecco da Silva Porto, L.E. Corrêa Antunes, Â. Diniz Campos Brazilian Agricultural Research Corporation, EMBRAPA, Brasília, Brazil. Key words: Colletotrichum spp., enzymatic activity, Fragaria x ananassa, Induced systemic resistance, K2HPO4. Abstract: The objective of this work was to evaluate a phytoprotective film of chitosan­pyroligneous extract in promoting growth, productivity, induction of systemic resistance in strawberry cultivars managed in an organic production system. Treatments consisted of rates (0, 25, 50, and 100 mL L­1) of Chi­Pyro­ Film and a reference resistance inducer (dipotassium hydrogen phosphate ­ K2HPO4), evaluated in three strawberry cultivars (‘Albion’, ‘San Andreas’ and ‘Portola’). Growth, yield, anthracnose incidence, and enzymatic activity were evaluated. The experimental design was a randomized block design with four replications. Chi­Pyro­Film increases the growth, yield, and anthracnose resis­ tance of strawberry plants. The best concentration of Chi­Pyro­Film varies between 50 and 60 mL L­1, according to strawberry cultivar. 1. Introduction Strawberry (Fragaria x ananassa Duch.) is the most planted red fruit in Brazil. The high demand is due to its sensory characteristics such as color, texture, aroma, and taste (Ventura­Aguilar et al., 2018). However, according to a report by the Brazilian Health Surveillance Agency (ANVISA) of 2016, 26% of strawberry samples collected between 2013 and 2015 presented nonconformities with pesticide residues. Pesticides detected not authorized for the crop, captan stood out, detect­ ed in 20.4% of the samples analyzed, among others such as dithiocarba­ mates, pyrimethanil, carbendazim, tebuconazole, iprodione, and azoxys­ trobin (ANVISA, 2016). Most of these fungicides aim to control or prevent anthracnose (Colletotrichum spp.), a disease that is considered main in strawberry fields in Brazil (Kososki et al., 2001; Capobiango et al., 2016). Currently, there is a search for technologies, which make agriculture sustainable and “smart”, with practices and ways to minimize the exces­ sive use of chemicals (Grewal et al., 2018). In this context, the use of resistance inducing products activates the plant’s natural defenses, enabling disease control in the organic production system. (*) Corresponding author: ivanspereira@gmail.com Citation: DOS SANTOS PEREIRA I., GRECCO DA SILVA PORTO F., CORRÊA ANTUNES L.E., DINIZ CAMPOS Â., 2022 ­ Phytoprotective film for resistance induction, growth, and yield of organic strawber‐ ries. ­ Adv. Hort. Sci., 36(1): 43­52. Copyright: © 2022 Dos Santos Pereira I., Grecco da Silva Porto F., Corrêa Antunes L.E., Diniz Campos Â. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 10 February 2021 Accepted for publication 25 February 2022 AHS Advances in Horticultural Science Adv. Hort. Sci., 2022 36(1): 43­52 44 The resistance is associated with various defense responses, such as protein and phytoalexin synthesis, cell wall changes, and increased activity of various enzymes defense­related (Durrant and Dong, 2004). Responses that are associated with changes in the activity of various enzymes such as peroxidase (POX, EC 1.11.1), polyphenol oxidase (PPO, EC 1.14.18.1), and phenylalanine ammonia­lyase (PAL, EC 4.1.3.5) (Prasannath et al., 2014; Prasannath, 2017). POXs have been implicated in many defense processes, such as hypersensitive response, lignifica­ tion, phenolic and glycoprotein cross­linking, suber­ ization, and phytoalexin production (Thakker et al., 2 0 1 2 ; P r a s a n n a t h , 2 0 1 7 ) . P P O s a r e a g r o u p o f enzymes that catalyze the oxidation of hydroxyphe­ nols to their quinone derivatives, which have antimi­ c r o b i a l p r o p e r ti e s ( P r a s a n n a t h , 2 0 1 7 ) . P A L (E.C.4.1.3.5) is the major enzyme in the phenyl­ propanoid pathway and acts in the synthesis of defense­related secondary compounds such as phe­ nols and lignins (Hemm et al., 2004; Vanitha et al., 2009). Among the compounds that have activating prop­ erties of defense mechanisms in plants are the chi­ tosan and pyroligneous extract (Di Piero and Garda, 2008; Grewal et al., 2018; Souza et al., 2018). Chitosan is a polycationic β­1,4 polymer bound to D­glucosamine chemically derived from crustaceans and soluble in organic acids and known to be a natur­ al elicitor and triggers various physiological and bio­ chemical responses in plants that act in the growth, production, and protection against disease (Chandra et al., 2015; Katiyar et al., 2015; Pichyangkura and Chadchawan, 2015). Chitosan has several characteris­ tics that make this polymer advantageous for many applications: (1) has a defined chemical structure; (2) may be chemically and enzymatically modified; (3) is p h y s i c a l l y a n d b i o l o g i c a l l y f u n c ti o n a l ; ( 4 ) i s biodegradable and biocompatible with many organs, tissues and cells; (5) can be processed into various products including flakes, powders, membranes, fibers and films (Badawy, 2012; van den Broek et al., 2015; Porto et al., 2019). The pyroligneous extract is a liquor with strong smoke flavors, is a crude and acid condensate pro­ duced from the distillation of the smoke generated in the carbonization of wood. It consists of a complex mixture of compounds derived from the chemical decomposition of wood components through the condensation of vapors and gases generated during pyrolysis in a low oxygen concentration (Campos, 2018; Pimenta et al., 2018). The pyroligneous extract is composed of water (80­90%) and more than 200 species of organic com­ pounds (10­20%) (Theapparat et al., 2018). The pres­ ence of phenolic compounds in the pyroligneous extract confers growth­promoting and antifungal p r o p e r ti e s , a s f o u n d i n t h e l i t e r a t u r e o n Helminthosporium sativum, Cochliobolus sativus, Waltz, Colletotrichum orbiculare, Alternaria mali (Jung, 2007; Baimark and Niamsa, 2009; Wei et al., 2010 a; Grewal et al., 2018). In this context, the cationic character of chitosan in acidic conditions offers the possibility to establish electrostatic interactions with other negatively charged compounds, for example with the pyrolig­ neous extract, considered a raw material obtained from renewable sources, and is a good solvent for chitosan (Campos et al., 2012; van den Broek et al., 2015; Porto et al., 2019). The phytoprotective film of chitosan­pyroligneous extract (Chi­Pyro­Film) consists of the chitosan dilut­ ed in the pyroligneous extract, and its characteristics are the formation of a film with photoprotection capability against radiation (UV­B and UV­C), fungi toxic action, and inducing systemic resistance in plants (Campos et al., 2012). The objective of the present work was to evaluate the effect of different concentrations of phytoprotec­ tive film formulated with chitosan and pyroligneous extract (Chi­Pyro­Film) and dibasic potassium phos­ phate (K2HPO4) on growth promotion, resistance induction to anthracnose, yield, and defense enzyme activity in strawberry cultivars in the organic produc­ tion system. 2. Materials and Methods The study was carried under field conditions, at Embrapa Temperate Climate Experimental Station, in Pelotas city, Rio Grande do Sul state, Brazil (31°40’49 “S 52°26’18” O, at 60 m altitude) (Fig. 1 a). The cli­ mate of the region, according to the Köppen classifi­ cation is Cfa type, temperate and humid, with hot summers. Raw materials The pyroligneous extract was obtained through an extraction procedure of Eucalyptus grandis proposed by Campos (2018) and the distillation process was performed according to that described by Porto et al. Dos Santos Pereira et al. ‐ Resistance induction in strawberries cultivars 45 (2019). Chitosan was supplied by Nutrifarm™, with a 97% degree of deacetylation, determined by proton magnetic resonance (Porto, 2011). Plant material The seedlings of neutral­day strawberry cultivars, ‘Albion’, ‘San Andreas’ and ‘Portola’, were planted in May and were grown under a low tunnel system with mulching and drip irrigation. The spacing between lines and between plants was 0.30 m, with three lines per bed. The area had a history of many years with severe anthracnose incidence in the strawberry plants (Fig. 1b, 1c). Liming and fertilization were per­ formed according to the recommendation for straw­ berry organic production. Climatic data from the experiment period are shown in figure 1d. Treatments The treatments of induction of systemic resis­ tance consisted of four concentrations of the Chi­ Pyro­Film and a reference treatment with dibasic potassium phosphate (K2HPO4). The Chi­Pyro­Film was registered in the field of Green Chemistry at the National Institute of Intellectual Property in Brazil (PCT/BR2013/000597), United States (US201503 36854A1) and Germany (DE112013006230T5) as a phytoprotective for agriculture use. The Chi­Pyro­Film with a concentration of 30 g L­1 (Fig. 2) was diluted with distilled water in different concentrations (0, 25, 50, and 100 mL L­1). Treatment with K2HPO4 was applied at a concentration of 50 mM (Orober et al., 2002; Aleandri et al., 2010). This compound has shown efficacy against e.g. powdery mildew on barley, cucumber, pepper, and tomato (Blumeria graminis f. sp. hordei, Sphaerotheca fulig‐ inea, Leveillula taurica, and Erysiphe oronti, respec­ tively), anthracnose (Colletotrichum lagenarium) on cucumber, rust (Puccinia sorgi) and leaf blight (Exserohilum turcicum) on maize and rice blast (Pyricularia oryzae), mildew (Sphaerotheca fuliginea) (Reuveni et al., 1996; Reuveni and Reuveni, 1998; Manandhar et al., 1998; Reuveni et al., 2000; Ehret et al., 2002; Orober et al., 2002; Hamza et al., 2017). The spraying of treatments started at the begin­ ning of fruiting, and the application dates were indi­ cated in figure 1d. The spray volume was 4.5 mL per plant (500 liters per hectare), applied through conical jet nozzles, observing a total coverage of the plants until close to the runoff point. Analysis of plant growth, damage by anthracnose, and fruit yield Vegetative growth evaluations consisted of dry mass (g plant­1) of crown, root, and leaf, by drying to constant weight (65°C) of three plants collected in each experimental unit at the end of the production cycle, 256 days after seedlings transplanting. The pro­ ductive variables measured were fruit yield (g plant­1) and fruit weight (g fruit­1), obtained by evaluating the total fruits harvested in each experimental unit. The percentage of fruits with anthracnose was obtained by counting the fruits with symptoms at each harvest (Fig. 1c). The harvests were carried out three times a week between October and January. Enzymatic activity analysis Biochemical evaluations were realized by determi­ nation of the specific activity of peroxidase (POX), Fig. 1 ­ Experiment under field conditions (a), anthracnose symptoms in strawberry plants (b) and fruits (c), and cli­ matic data from the experiment period (d). Fig. 2 ­ Electron micrograph of the phytoprotective film of Chi­ Pyro­Film, after spraying on a smooth surface at a tem­ perature of 18 to 25°C (a and b). Chitosan strawberry leaf covered with a film formed by Chi­Pyro­Film (c). Adv. Hort. Sci., 2022 36(1): 43­52 46 3. Results Chi­Pyro­Film concentrations had a significant effect on the growth, yield, anthracnose resistance induction, and enzymatic activity of strawberry plants. Growth and development The dry mass of the plant showed factors interac­ tion. The three cultivars showed a quadratic response to Chi­Pyro­Film concentrations (Fig. 3), but the high­ est efficiency concentration was different. ‘Albion’ and ‘Portola’ had the highest efficiency concentration estimated at 60 mL L­1 of Chi­Pyro­Film, while for ‘San Andreas’ the concentration was 50 mL L­1 (Fig. 3). The treatment with reference resistance inducer, K2HPO4, had also different according to cultivars. In ‘Albion’, it provided a leaf mass slightly higher than the control treatment and similar to the 100 mL L­1 Chi­Pyro­Film concentration, but it was lower than 25 and 50 mL L­1 (Fig. 3). The yield also was significantly influenced by Chi­ Pyro­Film, with a quadratic response to isolated effect of the film concentration factor (Fig. 4). Regardless of cultivar, the highest efficiency concen­ tration was estimated at 60 mL L­1. Regarding the ref­ erence treatment (K2HPO4), it was observed that it had a performance similar to 100 Chi­Pyro­Film and higher than control (0 mL L­1 of Chi­Pyro­Film), but less than the 50 mL L­1 (Fig. 4). Between cultivars Fig. 3 ­ Dry mass of leaves of Albion, Portola and San Andreas cultivars, in response to Chi­Pyro­Film concentrations and reference treatment (K2HPO4, 50 mM). Interaction effect between factors (cultivar and film concentration). *, **, ***, significant at p<0,05, p<0,01, p<0,001, respec­ tively. polyphenoloxidase (PPO), and phenylalanine ammo­ nia­lyase (PAL) enzymes, in strawberry leaf samples collected immediately before application (BA) and 48 hours after application (AA) of Chi­Pyro­Film concen­ trations and reference treatment (K2HPO4). For the extraction of POX and PPO, was used 500 mg of ground tissue below 4°C in 10 mL of 0.05 M phos­ p h a t e b u ff e r ( p H 7 . 0 ) c o n t a i n i n g 1 m g o f polyvinylpyrrolidone­10. Subsequently, centrifuga­ tion was performed at 4,000 g for 30 minutes under refrigeration. The supernatant was preserved on ice and used for determinations according to Campos et al. (2003). The POX and PPO extraction was carried o u t b y g r i n d i n g t h e l e a v e s w i t h 2 0 m g polyvinylpyrrolidone (Sigma­Aldrich). The enzyme extract obtained after filtration (Whatman 1) and centrifugation (5,600 gn, 15min) were used to test the activity. POX activity was determined in the enzyme extract mix with a phosphate­citrate buffer com­ posed of 0.2 M sodium phosphate solution and 0.1 M citric acid (pH 5.0). The mixture was homogenized in vortexed for 15 seconds. POX activity was determined according to Campos et al. (2004). PPO activity was determined in the enzyme extract with 3.6 mL of 0.05 M phosphate buffer (pH 6.0) and 0.1 mL of 0.1 M catechol. PPO activity was determined according to Campos et al. (2004). PAL activity was determined in crude leaf extracts according to the methods described by Hyodo and Yang (1971) and Hyodo et al. (1978) modified by Campos et al. (2003). For extraction, 500 mg of tissue was macerated (below 4°C) with 8 mL of 50 mM sodium borate buffer (pH 8.5) containing 25 g L­1 of polyvinylpyrroli­ done­10 and 4 mL L­1 of mercaptoethanol. The pro­ tein extract obtained after filtration (Whatman 1) and centrifuged (5,600 gn, 30 min) under refrigera­ tion (below 4°C). was used to assay the PAL activity. Protein in the extracts was determined by the Bradford method (Bradford, 1976). Experimental design and statistical analyses The experimental design was randomized blocks with four replications of nine plants. The results were submitted to variance analysis and the means of the variables with significant effect were compared using the Tukey test (cultivars) or regression analysis (con­ centrations) at 5% error probability. Dos Santos Pereira et al. ‐ Resistance induction in strawberries cultivars 47 effect, ‘Portola’ showed the highest yield than ‘Albion’ and ‘San Andreas’ (Table 1). Results similar to those observed by Carini et al. (2015) in a study of evaluation of strawberry cultivars in an organic sys­ tem, in which ‘Portola’ was also more productive than ‘Albion’ and ‘San Andreas’. The fruit weight was not influenced by Chi­Pyro­ Film treatments. However, there was an effect of the cultivar factor, with ‘San Andreas’ producing fruits of a higher mass (Table 1). Corroborating with Carini et al. (2015), which evaluated the same three cultivars, found a higher fruit weight of ‘San Andreas’, but with the same weight that ‘Albion’. The variable fruits with anthracnose showed an isolated effect of the factor Chi­Pyro­Film concentra­ tions, that is, all cultivars had the same response to the application of Chi­Pyro­Film, with a quadratic reduction in the number of fruits attacked, with the maximum efficiency concentration estimated at 60 mL L­1 Chi­Pyro­Film (Fig. 5). The reference treatment with K2HPO4 was similar to the 100 mL L ­1 of Chi­Pyro­ Film, but lower than the 25 mL L­1 and 50 mL L­1 (Fig. 5). Among the cultivars, ‘Portola’ was the most sensi­ tive to the occurrence of anthracnose in fruits, with no difference between ‘Albion’ and ‘San Andréas’ (Table 1). Enzymatic activity In the present study, there was a significant effect of Chi­Pyro­Film concentrations on the activity of POX, PPO, and PAL enzymes (Figs. 6 and 7). An inter­ action effect between film concentration and sam­ pling time indicated that 48 hours after application, there was a quadratic effect of film concentrations on the activity of the three enzymes studied. In the case of POX and PPO, an activity reduction effect was obtained up to the estimated concentrations of 54 mL L­1 and 50 mL L­1, respectively, followed by an increase (Fig. 6a and 6b). However, PAL activity increased until the estimated concentration of 36 mL L­1, with a subsequent reduction (Fig. 6c). About the Fig. 4 ­ Yield of strawberry, in response to Chi­Pyro­Film concen­ trations and reference treatment (K2HPO4, 50 mM). Isolated effect of film concentration (average of cultivars responses). *, **, ***, significant at p<0,05, p<0,01, p<0,001, respectively. Table 1 ­ Yield, fruit weight and fruits with anthracnose in Albion, Portola and San Andreas cultivars (z) (z) Means followed by different lowercase letters in the row, differ by Tukey test at 5% error probability. The averages correspond to the isolated effect of cultivar factor to studied variables. Variables Cultivar C.V. (%) Albion Portola San Andreas Yield (g plant­1) 131.90 ± 7.93 c 221.35 ± 12.87 a 170.4 5± 9.30 b 20.18 Fruit weight (g fruit­1) 8.60 ± 0.67 b 8.50 ± 0.57 b 13.33 ± 1.09 a 72.71 Fruits with anthracnose (%) 5.29 ± 1.71 b 9.13 ± 1.53 a 5.83 ± 1.49 b 107.57 Fig. 5 ­ Percentage of fruits with anthracnose in response to Chi­ Pyro­Film concentrations and reference treatment (K2HPO4, 50 mM). Isolated effect of film concentration (average of cultivars responses). *, **, ***, significant at p<0,05, p<0,01, p<0,001, respectively. 48 Adv. Hort. Sci., 2022 36(1): 43­52 reference treatment, with K2PHO4, it induced activi­ ties similar to Chi­Pyro­Film in the concentration of 100 mL L­1 for POX, PPO, and PAL. The enzymatic activity also had an interaction effect between treatments of resistance induction and strawberry cultivars. POX activity decreased to concentrations of 49 mL L­1 and 57 mL L­1 in Albion and Portola, respectively (Fig. 7a). For San Andreas, although there was a similar trend, it was not signifi­ cant (Fig. 7a). Fig. 6 ­ Specified activity of peroxidase­POX (a), polyphenoloxi­ dase­PPO (b) and phenylalanine ammonia lyase­PAL (c) in the leaves, before (BA) and after application (AA) of Chi­Pyro­Film concentrations and reference treatment (K2HPO4, 50 mM). Interaction effect between film con­ centration and sampling time (before and 48 hours after application). *, **, ***, significant at p<0,05, p<0,01, p<0,001, respectively. Fig. 7 ­ Specified activity of peroxidase­POX (a), polyphenoloxi­ dase­PPO (b) and phenylalanine ammonia lyase­PAL (c) in leaves of Albion, Portola and San Andreas, in response to Chi­Pyro­Film concentrations and reference treatment (K2HPO4, 50 mM). Interaction effect between film con­ centration and cultivars. *, **, ***, significant at p<0,05, p<0,01, e p<0,001, respectively. Dos Santos Pereira et al. ‐ Resistance induction in strawberries cultivars 49 The cultivars Albion and San Andreas also had a quadratic response to Chi­Pyro­Film, with a decrease in PPO activity up to the estimated concentrations of 54 mL L­1 and 55 mL L­1 (Fig. 7b). Concerning Portola, there was a linear increase up to a 100 mL L­1 concen­ tration of Chi­Pyro­Film (Fig. 7b). Concerning PAL, there was a quadratic response in Albion, with an increase up to the 38 mL L­1 rate of Chi­Pyro­Film, as well as a linear reduction in Portola (Fig. 7c). The reference treatment with K2HPO4, showed a response similar to the 100 mL L­1 film concentration for the enzymes PPO and PAL in the three cultivars studied (Figs. 7b and 7c). For the POX in Albion and San Andreas, the reference treatment was similar to the 25 mL L­1 concentration, while in Portola, it was similar to the 50 mL L­1 film (Fig. 7a). 4. Discussion and Conclusions In general, the results indicate that Chi­Pyro­Film contributed to the increase of vegetative growth and yield, as well as, to the anthracnose damage reduc­ tion in strawberry fruits. Results that are in agree­ ment with those found in the literature, where it is observed an increase in vegetative, productive, and health variables, such as height, number of leaves, leaf area, yield and reduction of incidence of diseases in plants that received chitosan or pyroligneous extract (El­Miniawy et al., 2013; Masum et al., 2013; Mungkunkamchao et al., 2013; Mukta et al., 2017; Kumaraswamy et al., 2018; Ventura­Aguilar et al., 2018). The effect of Chi­Pyro­Film on strawberry plant growth can be attributed to the role of chitosan as a non­toxic and biodegradable plant growth promoter (Salachna and Zawadzińska, 2014; Ahmed et al., 2020). Some authors suggest that foliar application of chitosan enhances the endogenous concentration of phytohormone such as gibberellic acid and auxin (Uthairatanakij et al., 2007; Ahmed et al., 2016). But the increase in macro and micronutrient accumula­ tion and improved the content of photosynthetic pig­ ments, provided by chitosan are also related to its influence on plant growth (Shehata et al., 2012; Ahmed et al., 2016). On the other hand, pyroligneous extract contributes to plant growth by its phytopro­ tective effect against pathogens, especially fungi. Was reported antipathogenic effects of the pyrolig­ n e o u s e x t r a c t o n p l a n t p a t h o g e n i c f u n g i l i k e Helminthosporium sativum, Cochliobolus sativus, Valsa mali, Colletotrichum orbiculare, and Alternaria mali (Jung 2007; Wei et al., 2010 a). This antifungal activity has been related to the presence of furalde­ hydes and phenols in pyroligneous extract (Grewal et al., 2018). The efficiency of spraying with Chi­Pyro­Film can be attributed to the effects of pyroligneous extract and chitosan individually, and to an interaction effect between both. According to Porto et al. (2019), who studied physicochemical properties of Chi­Pyro­Film, the film showed a semicrystalline structure, which is smooth and stable up to 50°C, being persistent in environmental conditions; it is permeable to water vapor and has high hygroscopicity, in addition to being able to efficiently block incident UVB and UVC radiation. The coverage presented by the Chi­Pyro­ Film, as well as its persistence on the leaf surface (Fig. 2), probably provide several days of action, per­ haps a large part of the application interval (15 days). The main effect of chitosan and the pyroligneous extract is attributed to resistance induction by increased defense enzyme activity and accumulation of phenolic compounds acting on reactive oxygen species (ROS) (Wei et al., 2010 b; Katiyar et al., 2015; Pichyangkura and Chadchawan, 2015; Grewal et al., 2018). This effect is in line with the behavior verified b y t h e P A L a c ti v i t y i n t h i s s t u d y ( F i g s . 6 c , 7 c ) . However, the activity of the POX and PPO enzymes responded differently, with a reduction in activity in the concentrations estimated between 50 and 60 mL L­1, 48 hours after application of treatments (Figs. 6a 6b), especially in the Albion and San Andreas culti­ vars (Figs. 7a, 7b). This film rate (50­60 mL L­1) had a better performance in the dry mass accumulation, yield, and incidence of anthracnose. T h e r e s u l t s i n d i c a t e t h a t i n a d d i ti o n t o t h e increase in systemic resistance, suggested by the PAL response, there is a direct effect of phytoprotection and a reduction of stress condition. Some aspects that can be associated with this second aspect, maybe the block that the film exerts concerning UVA and UVB radiation, as well as its potential action on pathogens, a hypothesis that corroborates the results obtained by Porto et al. (2019). In this way, it can be suggested that Chi­Pyro­Film has a complex performance, acting both on metabo­ lism with increased plant resistance and reducing cel­ lular damage caused by physical (radiation) and bio­ logical stress (pathogens), as well as forming a phyto­ protective film that inhibits the direct action of stress Adv. Hort. Sci., 2022 36(1): 43­52 50 agents, such as UVA and UVB radiation and inhibiting the attack of pathogens. In general, Chi­Pyro­Film showed greater efficien­ cy in promoting growth, yield, and resistance to anthracnose than the reference treatment (K2HPO4), mainly in concentrations between 25 and 50 mL L­1. K2HPO4 performed similarly to the 100 mL L ­1 of the film. Different studies indicate the effect of K2HPO4 in the resistance induction of several species (Reuveni and Reuveni, 1998; Kashiap and Dhiman, 2009; Aleandri et al., 2010; Hamza et al., 2017; El­Tanany et al., 2018). According to Orober et al. (2002), foliar application of K2HPO4 results in the activation of sys­ temic resistance mechanisms. The positive effect of K2HPO4 is associated with salicylic acid involved in t r i g g e r i n g p l a n t c e l l d e f e n s e a n d s e n s i ti z a ti o n responses for a faster and stronger response to sub­ sequent pathogen attack (Mauch­Mani and Métraux, 1998; Orober et al., 2002). The 100 mL L­1 concentration of Chi­Pyro­Film may b e h i g h f o r t h e s t r a w b e r r y c r o p . P r o b a b l y t h e increase in film thickness, which according to Porto et al. (2019), can significantly reduce the film’s per­ meability to water vapor. An aspect that can make some physiological processes such as the flowing water, the absorption of nutrients, and photosynthe­ sis less efficient. This study provides indicates that the phytopro­ tective film (Chi­Pyro­Film) is effective as a growth promoter and inducer of systemic resistance to anthracnose, resulting in increased growth and fruit production in strawberry plants of different cultivars. The optimal concentration of Chi­Pyro­Film ranges between 50 and 60 mL L­1, depending on the straw­ berry cultivar. We believe that the tested film can be an impor­ tant tool for production systems, especially agroeco­ logical and organic systems, which require alterna­ tives for disease control. But also, due to its physical­ chemical properties and great stability, we believe that the film can be combined with other compo­ nents, such as nutrients, biostimulants, and plant hormones, to enhance its effect. However, such com­ binations need to be studied. 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