Control of fungal spoilage in strawberries using crude plant extracts against the fungus Botrytis cinerea

Abstract The study evaluated the conservation of strawberries treated with crude plant extracts (barbatimão, sibipiruna, guarana, and catuaba) against fungal deterioration and physicochemical characteristics. MIC of 0.125; 0.0156; 0.25 and 0.0312 g/mL were found for barbatimão, sibipiruna, guaraná and catuaba, respectively, against B. cinerea . Treated samples showed no fungal deterioration during 11 days. Analyzes of weight loss, soluble solids, titratable acidity, and pH variation were performed. Sibipiruna showed lower values of mass loss, and the greatest occurred for the catuaba extract. Barbatimão did not change soluble solids and stood out with catuaba in the color parameters L and a*. Small changes in pH were observed with time. Soluble solids maintained values between 6.47 oBrix and 9.90 oBrix for catuaba and sibipiruna extracts at zero and six days. Principal component analysis did not show a strong correlation between the variables. The extracts become alternatives for strawberry conservation, increasing conservation and maintaining physicochemical characteristics. Graphical Abstract


Introduction
Strawberry is a bright red, juicy, fragrant fruit obtained from the strawberry plant, with a sweet taste and nutritional values. Is widely consumed fresh and processed as preserves, jams, or as a component of yogurts and ice creams (Barrazueta-Rojas et al. 2018;Siedliska et al. 2018;Dos Santos et al. 2019;Bortolini et al. 2022). Has high metabolic activities and high moisture content, sugars and acids being susceptible to degradation by microorganisms, such as B. cinerea (Jia et al. 2016;Roca-Couso et al. 2021), the causative agent of gray mold, is the primary necrotrophic pathogen that potentiates plant tissue damage (Herrera-Romero et al. 2017;Petrasch et al. 2022).
B. cinerea is responsible for infecting many plants, mainly in the post-harvest period and produces toxins and enzymes capable of degrading the cell wall, which induces necrosis of plant tissues, favoring the infection process (Shao et al. 2021). The infection begins in the flowers through wounds present on the surface of the fruit (Husaini et al. 2012;Petrasch et al. 2022). It can survive saprophytically and the dispersal of conidia through air and water affects susceptible crops (Palaretti 2018). It is genetically variable, acquiring resistance to chemicals to control it. Infection caused by the fungus in fruits causes economic losses (Sansone et al. 2018). Thus, for gray mold disease, techniques such as the application of natural compounds and physical treatments are used as an alternative measure (Freitas et al. 2015).
Poincianella pluviosa (sibipiruna), cultivated in a tropical climate, it has small, deciduous leaves like the foliage of Pau-Brasil (Sharma et al. 2017;Vieira Fantaus Pinto et al. 2021). Has a significant source of bioactive metabolites and bioflavonoids, steroids, and others, which help in antimicrobial, anti-inflammatory, anti-rheumatic, and anti-rheumatic analgesic activities (Kayano et al. 2011;Domingos et al. 2019).
Stryphnodendron adstringens Beth (barbatimão) is a tree found on the coast and in the Brazilian cerrado, in which the chemical constituents are mainly tannins, but also present resins, essential oils, mucilages, and red coloring matter. Tannins have a polyphenolic composition and act against insects (Fonseca et al. 2018;Soares et al. 2022).
It has already been used in other ways, such as to treat leucorrhoea, diarrhea, and gynecological infections (Valera et al. 2013;Nascimento et al. 2021).
Paullinia cupana (guaran a), has large, dark green leaves and can be marketed in the form of vines with roasted grains, in the form of syrup, and even as a powder (Carvalho et al. 2016;Souza et al. 2017;Ruchel et al. 2021). De Faria Neto et al. (2019 state that the seeds have antioxidant and energetic properties, as they present a percentage of caffeine and tannin. Ventura et al. (2018) state that the dry extract of guarana is composed of theophyllines, theobromines, starch, potassium, calcium, phosphorus, iron, vitamins, and sugars, and has antimicrobial (Ruchel et al. 2021).
Trichilia catigua (catuaba) is found in semideciduous forests and part of the Atlantic Forest and is characterized by a thin, smooth, bitter, and astringent bark (Pizzolatti et al. 2002;Bernardo et al. 2021). This plant consists of condensed tannins, epicatechin and steroids, and hydrolyzable saponins, with an inhibitory activity on bacterial growth (Longhini et al. 2017;Panizzon et al. 2019).
These natural compounds mentioned above could act, theoretically, in inhibiting microbial growth, being a potential use in the conservation of fruits, such as strawberries.
The use of alternatives that complement the low temperatures for fruit conservation is important, due to the high content of water and sugars, which limits the postharvest life of these foods (Assis and Britto 2014;Duan et al. 2019). Given these considerations, the objective of this work was to evaluate the conservation of strawberries with the raw plant extracts of barbatimão, sibipiruna, guarana and catuaba against fungal deterioration.

Minimum inhibitory concentration
The control samples of control and treated strawberries with the alcoholic solutions of each extract were visually checked each day after treatment to verify the beginning of fungal deterioration, using an 11-day limit for observation. Treated strawberries showed no visible deterioration in 11 days of treatment for all extracts, compared to the control, which showed the beginning of deterioration on the third day. The treatments that kept the strawberries preserved for the longest time, without evidence of deterioration, were those with triple MIC values. These strawberries remained intact in visible fungal decay during the established period. The results found for the MIC of the extracts against the fungus Botrytis cinerea are shown in Table 1S, in which the sibipiruna extract showed the highest inhibition power, followed by the catuaba, barbatimão, and guarana extracts.
Sibipiruna extract obtained the lowest MIC, equal to 0.0156 g/mL, necessary to inhibit B. cinerea. Andriani et al. (2021) showed antifungal activity of the ethyl acetate fraction (FAE) from the stem bark of Poincianella pluviosa in the planktonic and biofilm mode of the pathogen C. neoformans, where they reported MICs that varied for planktonic and sessile cells, showing antifungal results (Andriani et al. 2021).
P. pluviosa extracts are related to antimalarial activity (Kayano et al. 2011), antifungal, anti-inflammatory, analgesic and antibacterial activity inhibiting Gram-positive and Gram-negative bacteria (Zanin et al. 2019). The ethanolic extract of sibipiruna leaves showed antifungal activity against three Colletotrichum isolates by reducing the mycelial growth rate, inhibiting sporulation, in some concentrations, above 60% (Duan et al. 2019).
The catuaba extract showed a MIC for B. cinerea, equal to 0.25 g/mL. Studies of T. catigua extract showed activity against vancomycin-resistant Enterococcus faecium (VREfm) and against strains of Staphylococcus aeruginosa, Escherichia coli, Pseudomonas aeruginosa, Salmonella Typhi, Shigella boydii, Shigella flexneri and Shigella dysenteriae with MIC varying by microorganism (Konat e et al. 2015;Ritter et al. 2021).
Methanolic extract from the fruit of Trichilia emetica showed activity against Candida albicans, Cryptococcus neoformans, Aspergillus flavus, Tricophyton mentagrophytes and Tricophyton violacium (Andriani et al. 2021). Sesquiterpenes isolated from Trichilia showed inhibitory activity for peroxidation in lipid membranes in mitochondria and antibacterial action (da ). Catuaba has alkaloids, tannins, bitter substances and resins responsible for antimicrobial activities (de Oliveira et al. 2009). Maria P ovoa Violante et al. (2012) highlight the protective action against lethal infections and antiviral activity in assays with catuaba extracts.
The crude vegetable extracts of barbatimão and guarana showed higher MIC values, equal to 0.125 g/mL and 0.25 g/mL, respectively. According to Reddy (2015), among the chemical constituents present in barbatimão, tannins are present in the majority, between 25% and 30% of the complexes with proteins; this element has fungicidal and bactericidal activity. The bark extract of this plant reduced the incidence of the fungus Fusarium subglutinans in pineapple (Reddy 2015).
Barbatimão demonstrated antifungal activity against Candida albicans, C. neoformans and Trychophyton rubrum, which has been attributed to condensed tannins that affect the wall of microorganisms, inhibiting the formation of the germ tube (Ishida et al. 2006;Ferreira 2013;de Freitas et al. 2018;Nabila and Putra 2020). The antibacterial activity of barbatimão hydroalcoholic extract against Streptococcus mitis and Lactobacillus casei has also been reported in studies ).
Guarana has a high content of caffeine, alkaloids, tannins and phenolic compounds that provide natural protection against fungi and bacteria (Marques et al. 2019). Studies have shown that guarana extract had inhibitory effects on fungal growth, decreasing colony diameter, antibacterial activity against Gram-positive (B. subtilis and S. aureus) and Gram-negative (E. coli and P. aeruginosa) bacteria (Antonelli Ushirobira et al. 2007;Maristela et al. 2014;Barrazueta-Rojas et al. 2018;de Freitas et al. 2018;Siedliska et al. 2018;Bortolini et al. 2022).

Mass loss
Based on Table 2S, it is possible to verify a significant difference between the weight loss of strawberries for the control, sibipiruna, guarana, catuaba, and barbatimão extracts at 0, 3, 6, and 9 days, independently. The most significant loss happened in the time of 9 days in the control sample and the extracts of sibipiruna, guarana, and barbatimão. Although there was no significant difference between times 6 and 9 days, time 6 was the one with the most significant loss for catuaba extract.
Comparing the extracts and the control sample, the greatest loss of mass occurred for the catuaba extract (7.54%) at time 6, and the smallest was for the sibipiruna extract. (0.63%) within 3 days ( Figure S1). There were always significant differences between the means and extracts over the 9 days, where the samples showed increasing mass loss.
According to Aitboulahsen et al. (2018), who evaluated the quality of post-harvest strawberry coated with gelatin combined with Mentha pulegium essential oil, the mass loss increases throughout storage making it independent of the treatments applied. Likewise, the less post-harvest handling the strawberries receive, the less mass loss will be since the loss occurs due to the loss of water vapor from the fruits to the external environment. Borges et al. (2013) declare that even the coatings are based on polysaccharides or proteins, with the effect of maintaining moisture and prolonging the shelf life, the components of these coatings have a hydrophilic nature that affects the formation of a desirable layer. Silva et al. (2015) worked with biofilms of yellow passion fruit residues applied to apples, finding that the coatings did not prevent the mass loss but showed lower values than when compared to the control, untreated strawberry. Mazaro et al. (2013) explain that the loss of mass in strawberries occurs due to the loss of water from the fruits through the processes of transpiration and respiration. As the strawberry has a thin epidermis and high moisture value, this fruit has significant mass losses. In this work, it was observed that all extracts showed excellent weight loss, but the sibipiruna extract was the one that differed the least from the control.
Thus, at all times and extracts, there were significant differences between the means and over the days and the difference in mass loss between the extracts, with catuaba with more significant loss and sibipiruna with less mass loss. Dotto et al. (2014) found that even using alcoholic extracts based on C. officinalis did not interfere with the mass loss values. However, with the extract in post-harvest strawberries, at all concentrations, there was a reduction of the fungus B. cinerea and obtaining total inhibition at doses above 2.5%.

Soluble solids and pH
The pH obtained significant differences in the extract at 0 and 3 days for barbatimão and catuaba extracts. In all extracts and the control sample, the alcohol showed a low pH value (<4.0) ( Table 2S). The highest value was for barbatimão extract (3.88) at 9 days, and the lowest was for guarana extract (3.42) at zero time. There were no significant differences between the samples between times 0, 6, and 9, as seen in Figure 2S.
According to Campos et al. (2014), who studied the postharvest quality of strawberries treated with pomegranate peel extracts, the pH analysis showed that the sprays showed slight differences, with pH values equal to 3.64 and 3.57, with values similar to 3.64 and 3.57. below 4 as found in this study.
The determination of pH in fruits helps in the control of microbial growth because, in the post-harvest period, the microorganisms require variables of pH, temperature, oxygen, nutrients, and humidity to develop; that is, depending on the acidity of the food, chemical reactions will take place (Qadri et al. 2015). F evrier et al. (2017) state that the decrease in pH is related to increasing other acids, increasing pulp acidity, and reducing pH. And consumers prefer to consume strawberries with a low acid pH; even these fruits tolerate a more acidic pH. Minarelli et al. (2014) evaluated strawberry fruits submitted to hydroxyethylcellulose and aqueous propolis application. Their results showed that, even with a difference between days of storage, the pH values varied little and did not interfere with the quality of the product. Analyzing the use of an active modified atmosphere and refrigeration in strawberries, the average pH values remained at 3.75, very close to the values found in this experiment.
In soluble solids, only the control and barbatimão extract had no significant changes over the nine days (Table 2S). Franc¸oso et al. (2008) indicate in their studies that depending on the variety of strawberries, the value of soluble solids presents different intervals. That is, the Toyonoka variety presents 9.07 to 10.60 o Brix, while the Dover and Campineiro varieties obtain 5.4 and 6.0 o Brix. The author Kader (1999) establishes that the ideal minimum value for soluble solids is 7.0 o Brix and in this work, only for the control extracts, catuaba and barbatimão presented values below 7.0 o Brix, on days 3, 6, and 9, respectively. Soluble solids maintained values between 6.47 o Brix and 9.90 o Brix for catuaba and sibipiruna extracts at zero and 6 days, respectively, as shown in Figure S3.
The soluble solids content indicates the number of sugars present in the fruit, considering other compounds, such as acids, vitamins, amino acids, and pectins (Fonseca et al. 2018). Franc¸oso et al. (2008) explain that soluble solids typically increase according to fruit maturation, either by biosynthesis or by degradation of polysaccharides. And in his work to evaluate the physicochemical changes in irradiated strawberries, he found that the soluble solids content was between 7.0 and 9.5 o Brix. Such values are very similar to the results of this work that between the 9 days of analysis, only the sibipiruna and catuaba extracts showed a high value equal to 9.59 o Brix and 9.66 o Brix, respectively.
In the experiment by Herrera-Romero et al. (2017), the lowest value of soluble solids was observed in the treatment with Trichodermil product, with a value equal to 6.34 o Brix. This occurred where the severity of the disease was lower since the higher the severity for B. cinerea, the higher the o Brix value due to the dehydration of the fruit due to the infectious process. Thiel et al. (2021) studied the quality of strawberries packed with collagen-based film with the addition of pink pepper essential oil and observed a significant increase in the soluble solids content in samples packed in FCC film (collagen fiber film) due to a more significant weight loss of the samples that caused a concentration of the soluble solids.
According to Campos-Requena et al. (2017), strawberries stored in starch films with the addition of essential oil and the control showed no significant differences. Both had a large increase in the content of soluble solids. Such results are similar to the values of this study. The crude plant extracts did not show significant differences over the nine days and the high importance of the mass loss, which may be one of the reasons for the significant increase in the soluble solids content.

Titratable acidity
For the titratable acidity values in Table 2S, the highest value observed was at time 0 for the sample of guarana and barbatimão extracts. For the control and sibipiruna and catuaba extracts, the highest acidity values were found on the sixth, third and ninth day, respectively. The lowest acidity was found on the ninth day for the control and extracts of barbatimão and guarana.
It is known that titratable acidity is the amount of acid present in a sample that reacts with a basic solution of pre-established concentration (Ferreira et al. 2020). So, among the values determined by the analysis of titratable acidity, the highest value identified at time zero was for barbatimão (1.12 g/100g) and the lowest value for the same extract (0.51 g/100g) in 9 days, with significant differences between the means only at the time of 9 days for samples of barbatimão and guarana ( Figure 4S).
According to the author Kader (1999), the optimal maximum value for titratable acidity of strawberries is the value of 0.80% citric acid. However, most of the importance of the crude plant extracts are above 0.80% of citric acid during the 9 days, and the samples with sibipiruna extract showed all values above 0.86% of citric acid. Almeida et al. (2019) researched alternative treatments in the post-harvest of organic strawberry, using the treatment of types of water, such as ozonated and electrolyzed alkaline, performing the analysis of titratable acidity and obtained. As a result of a significant difference between the treatment and time interaction, all averages remained above 0.90%, the recommended value.
According to Ganhão et al. (2019), the acidity content of fruit indicates the state of conservation, as the acid decreases the growth of microorganisms and the action of degradation enzymes. Likewise, Moritz et al. (2021) state that pH and titratable acidity, in addition to determining fruit deterioration, also involve enzyme activities, texture, taste, and odor retention.
The values obtained for titratable acidity in samples of minimally processed strawberries kept under a controlled atmosphere at 5 and 10 C and 85%RH ranged from 0.55 to 0.65 in work by Moraes et al. (2008). They submitted strawberries minimally processed and kept under refrigeration and a controlled atmosphere. The use of atmospheres containing 3% O 2 þ 10% CO 2 and 3% O 2 þ 15% CO 2 was essential for maintaining the quality of minimally processed strawberries. They maintained better firmness ambient atmosphere more effectively in controlling fruit mass loss.

Color parameters
The L Ã parameter represents the luminosity/brightness of the analyzed surface. The closer to zero this parameter is, it indicates that the color is closer to black, and, in this case, the closer to 100, it suggests a color closer to white. For fruits, the lower the L Ã for a skin analysis, the greater the probability that the fruit has not reached the required degree of maturity for consumption, emphasizing that it has a greener and darker color (Motta et al. 2015). Regarding this parameter, the brightest sample (52.25) was found in the control sample on the ninth day and the darkest in the guarana extract (33.28) at the time of 3 days, as shown in Figure 5Sa. Thus, it can be said that there was a darkening of the strawberry coated with guarana extract and that naturally, there was a darkening over the 9 days, a result also observed by Aamer et al. (2021) by placing green tea, grape skin and rosemary extracts in strawberry nectar. Regarding the parameters a Ã and b Ã , the highest value (48.46) was determined at time zero for the catuaba extract and the lowest value (11.59) for the guarana extract at 6 days for the parameter a Ã . Higher (47.12) and lower (8.43) values of the parameter b Ã were verified in the catuaba extract at the time zero and sixth day, respectively, as shown in Figures 5Sb and c. A decrease in the a Ã parameter and an increase in b Ã would mean that the strawberries would be turning yellow, which did not happen in any of the samples. On the other hand, the higher the values of the a Ã parameter, the redder the strawberries, as Colussi et al. (2021) verified by encapsulating strawberries using allyl isothiocyanate electrospun ultrafine zein fibers. Figure 6S presents a principal component analysis (PCA) for the physicochemical evaluation of the control samples and the four extracts at 0, 3, 6, and 9 days. In Figure  6Sa, in which observations were made at time 0, the first (PC1) and second (PC2) dimensions explained 77.84% of the total variation. Main component 1 (PC1) represented 55.98%, while main component 2 (PC2) was 21.86%. In Principal Component Analysis (PCA), variables are represented as vectors, which characterize the samples located close to them. The longer the vector, the better the explanation of the variability between samples, and the values obtained by Pearson's correlation confirm the relationship between the parameters observed in the principal component analysis (Table 2S (a)), demonstrating correlations between some variables studied at time 0 The mass loss has a negative correlation with pH (-0.02366), soluble solids (-0.14980) and the color L Ã parameter (-0.37647) and buoyant with the titratable acidity (9. 48486) and with parameters a Ã (0.50197) and b Ã (0.49655). It is worth mentioning that there was no loss of mass at time zero for the samples.
It is also possible to observe in Figure 6Sa that higher values of titratable acidity and parameters a Ã and b Ã are found for the sample in which catuaba extract was added. In comparison, higher L Ã and pH values were found for the samples control and added with sibipiruna extract. Higher values of total solids are present in the sample composed of guarana.
In Figure 6Sb, in which observations were made for the third day, the first (PC1) and second (PC2) dimensions explained 78.08% of the total variation. Principal component 1 (PC1) represented 59.99%, while principal component 2 (PC2) was 18.09%. The relationship between the parameters observed in the principal components analysis (Table 3S (b)) demonstrates correlations between some variables studied on the third day. It is worth mentioning that the mass loss began to undergo variations and presented a negative correlation with pH (-0.00229), titratable acidity (-0.44797), and the color L Ã parameter (-0.43685) and positive with soluble solids (0.40665) and parameters a Ã (0.46386) and b Ã (0.46252). The most significant mass loss occurred for the sample with sibipiruna, and the highest pH value was for the control sample, both inversely proportional. Higher values of total solids and parameters a Ã and b Ã occurred for the sample with guarana, and the sample composed of catuaba had higher values of L Ã and titratable acidity. The physicochemical parameters were inversely proportional for the two samples in these two cases.
For the times of 6 and 9 days, there was an explained variation of 82.18% (PC1 ¼ 49.34% and PC2 ¼ 32.84%) and 89.66% (PC1 ¼ 64.59% and PC2 ¼ 25, 07%), respectively (Figure 6Sc and d). On the sixth day of treatment, it is possible to verify in Table 3S (c) and (d) that there is a quadrant inversion for mass loss. While at times 0 and 3, it was in the positive quadrant of PC1, it moved to its negative quadrant at times 6 and 9 days. It positively correlated with the color parameters at times 6 and 9 and only a negative correlation with soluble solids (-0.41407) at time 9. The highest loss of mass, titratable acidity, and soluble solids were determined for the sample with guarana at a time of 6 days. Higher pH for catuaba, inversely proportional to parameters a Ã and b Ã .
It is important to note that at no time was there a strong correlation between the variables, either positively or negatively, because when two variables are perfectly positively correlated (r ¼ 1), they essentially move in perfect proportion in the same direction while two sets that are perfectly correlated negatively impact in perfect proportion in opposite directions.

Conclusion
The minimal inhibitory concentration analysis confirmed the antifungal action of crude plant extracts against the filamentous fungus and phytopathogen Botrytis cinerea CCT 1252. Among the extracts, sibipiruna showed greater inhibitory power over MIC. In addition, when applied to strawberries and stored at 10 C, the extracts were kept the same, preserved for at least 8 days longer than untreated strawberries.
Regarding the physicochemical analyses, the extracts showed slight differences in the control but with values by limits already established in other studies. Among the extracts, the strawberries treated with sibipiruna had the lowest mass loss, remaining within the values for both pH and soluble solids. Only in the analysis of titratable acidity the barbatimão extract comes closer to the control. In the color parameters, the treated samples showed a darkening comparable to the control, without loss of reddish color, or tendency to yellowing, according to parameters a Ã and b Ã . Principal components analysis showed no strong correlation between the variables. Therefore, it can be concluded that the plant extracts applied to strawberries increased the storage time under refrigeration, maintaining physicochemical characteristics and inhibiting B. cinerea, the primary spoilage fungus and, compared in general the extracts, the strawberry samples treated with crude extract of sibipiruna stood out, presenting the best inhibitory concentration to the fungus and values of mass loss, titratable acidity, soluble solids and color parameters close to untreated strawberries. In this way, the use of these extracts becomes promising as preservatives for strawberries and other fruits attacked by the same fungus, acting as an alternative to control deterioration in the post-harvest period and helping to understand the antifungal activity in filamentous fungi by plant extracts. Future studies using fractions and synergism of extracts may be carried out, aiming at even more effective results.