Investigating the tolerance of different strawberry cultivars to Botrytis cinerea infection and its relation with fruit quality

Abstract

BACKGROUND:

Strawberries are a delicate, high nutritional value fruit with an extremely short shelf life and high susceptibility to tissue infection, mainly by Botrytis cinerea. Control of the disease requires an extensive amount of fungicide that is applied in varying complexes because the pathogen easily develops resistance against the active compounds. Planting tolerant cultivars seems to be a promising alternative for fruit growers, but there are currently no cultivars available combining tolerance to B. cinerea with attractive horticultural traits.

OBJECTIVE:

In this study, four well-defined strawberry cultivars (‘Romina’, ‘Cristina’, ‘Silvia’, and ‘Sibilla’) were selected and tested under treatment with Botrytis cinerea to determine the tolerance of each cultivar and its association with fruit quality.

METHODS:

Fruits were harvested and then stored for four days after inoculation (treatment) or not (control) with spores of B. cinerea; then, every day the level of decay was detected, and fruits were frozen for successive evaluation of fruit quality.

RESULTS:

The ‘Silvia’ cultivar is the one that demonstrated a higher level of tolerance to Botrytis infection during the treatment, and the ‘Romina’ cultivar was the cultivar most susceptible to the treatment. The results of the study also highlighted qualitative changes in all strawberry cultivars infected with Botrytis cinerea, by a decrease in the soluble solids content and an increase of acids. Generally, in all cultivars, phenolic acids, and vitamin C decreased in both control and infected but there was a strong decrease in infected fruit compared to control. Anthocyanin content increased in control fruits but strongly decreased in infected.

CONCLUSION:

As a preliminary outcome, a positive correlation was obtained between fruit nutritional quality and tolerance to decay, as a high amount of nutritional compound content provides lower susceptibility of fruits to Botrytis cinerea. To confirm this result more studies need to be done.

Keywords

Anthocyanins postharvest polyphenols vitamin C soluble solids

1 Introduction

Strawberry is one of the most cultivated and widespread fruits in the world and represents an important fruit tree crop: the cultivation of strawberries worldwide covers 397,000 ha, with a production that exceeds 9.5 million tons in 2022 [1]. Most of the production is concentrated in China and the United States, which together account for about 50% of the total world production. Europe remains one of the main production areas, where most of the production is concentrated in Spain and Italy.

Strawberries have been part of the human diet for centuries and they are considered a very rich dietary source of bioactive compounds (vitamin C and B9) and phenolic compounds (phenolic acids, flavonoids, and anthocyanins) [2 –9].

Strawberry is a highly perishable fruit, characterized by a short shelf-life, which can be affected by numerous pathogenic species, such as fungi, bacteria, viruses, and nematodes. The onset of the illness can result in a decline in the product’s commercial grade, a variety of damages, and, in the worst scenarios, even the death of the plant.

One of the main strawberry pathogens is Botrytis cinerea, the causal agent of grey mold, followed by Rhizopus stolonifera, Mucor spp., Colletotrichum spp., and Penicillium spp. [10]. These pathogens cause postharvest diseases as a result of latent infections that begin in the growing season in the field and infections from wounds sustained during harvest and handling operations [11]. Early detection of fungal-infected fruit is very important both for producers and consumers. Fungal contamination is especially dangerous in packing houses during storage, transport, and marketing procedures because even a very small number of infected fruits can spread the infection to adjacent healthy strawberries. The presence of fungal diseases on fruit surfaces not only causes a loss of quality but also diminishes the safety of the final product. Some fungal genera and species can produce mycotoxins, which cause infections or allergies in susceptible individuals [12].

In modern production, one of the most common management practices used for the prevention of postharvest rot is the use of fungicides, which are applied several times on the canopy of strawberry plants. However, the concern related to the presence of residues by the population, the growing resistance of the fungus, and the legal restrictions related to the use of chemicals, have led to advanced studies to find new alternatives. The use of ecological treatments and breeding activity are some examples that aim at a reduction in the use of fungicides and an increase in plant tolerance to the fungus. Numerous studies are finalized to identify the best less susceptible cultivars on the market and experiment with new tools that can allow the early identification of the disease directly in the field.

Future research does not exclude the possibility of genetic modification and breeding techniques to produce commercial cultivars characterized by a higher B. cinerea tolerance. Furthermore, the growing attention on qualitative and nutritional parameters, such as fruit sugar, acidity, and bioactive substance content, has led to an in-depth study of the cultivars affected by the disease. Some studies are focused on identifying tolerance to Botrytis disease and how this response can be correlated with the content of polyphenols, anthocyanins, and other fruit nutritional and qualitative compounds [10 , 14]. This is to identify new cultivars with increased tolerance to the disease combined with higher fruit quality.

For the present study, four well-defined strawberry cultivars (‘Romina’, ‘Cristina’, ‘Silvia’, and ‘Sibilla’) were selected and tested under treatment with Botrytis cinerea, the fungus that causes grey mold. The aim is to determine the nutritional and qualitative changes, evaluate the content of soluble solids, titratable acidity, vitamin C, anthocyanins, and phenolic acids, and inquire if these fruit quality parameters are related to the response and tolerance of each cultivar to the disease caused by the fungal pathogen.

2 Materials and methods

2.1 Plant materials

Following the method outlined by Mezzetti et al. [15], strawberries were grown at the “P. Rosati” Experimental Farm (43°31’N 13°36’E. 46 m altitude), in Agugliano (Ancona, Italy), at non-fumigated soil with the following main characteristics: pH 7.9, active calcium 9%, and texture composed at 40% clay, 25% sand, and 35% silt. The qualitative parameters and response to Botrytis disease were evaluated on four strawberry cultivars: ‘Cristina’, ‘Silvia’, ‘Sibilla’, and ‘Romina’.

The strawberry harvest took place between the end of April and the end of June 2020, selecting only the fruits that have reached commercial ripeness from each genotype, characterized by a uniform red color, absence of damages that could alter their conservation, and typical firmness of the cultivar. A total of 108 fruits were sampled for each cultivar: the fruits were selected from various plots and collected during the third and fourth harvests.

2.2 Inoculation and damage assessment from Botrytis cinerea

For evaluating the tolerance of different cultivars to Botrytis cinerea, the protocol indicated by Siedliska et al. was used [16]. Before the inoculation procedure, Botrytis cinerea was grown on agar for two weeks at room temperature. The fungal conidia were scraped from the surface of the agar with a sterile glass rod in a small volume of sterile water and diluted to a concentration of about 10⁶ spores mL^–1. During the inoculation, strawberry fruit of the different cultivars were immersed in Botrytis cinerea (10⁶ spores mL^–1) or sterile distilled water for the control, for 30 seconds, and dried at room temperature under an air cabin. During the whole experiment (4 days), control and inoculated fruit samples were stored in dark chambers at 20±1 °C and 90% relative humidity (RH), according to the procedure proposed by Wang et al. [17] and Guidarelli et al. [18]. An empirical scale of six degrees was used to measure the severity of the decay: 0 healthy fruit; 1 fruit with a surface infection of 1% to 20%; 2 fruit with a surface infection of 21% to 40%; 3 fruit with a surface infection of 41% to 60%; 4 fruit with a surface infection of 61% to 80%; and 5 fruit with a surface infection of 81% and sporulation (Fig. 1). The decay McKinney index was calculated using the formula I% = [Σ((d×f))/((N×D))]*100, where d is the category of rot intensity scored on the fruit, f is its frequency, N is the total number of fruit examined (both infected and healthy), and D is the highest category of decay intensity that occurred on the empirical scale [19]. After each day, evaluated fruits were stored at –80°C for other sensorial and nutritional parameters analysis.

Fig. 1

Botrytis cinerea susceptibility trial in fruits of ‘Cristina’ (A) control at 0, (B) control at day 4 (C) infected fruit at day 0, and (D) infected fruit at day 4.

2.3 Soluble solid contents and Titratable acidity

Soluble Solids (SS) were determined using a hand-held refractometer (ATAGO), and results are expressed as ° Brix. Titratable Acidity (TA) was determined from 10 mL of juice diluted with distilled water (1/2 v/v) and titrated with 0.1 N NaOH solution, until pH 8.2, and expressed as mEQ of NaOH per 100 g Fresh Weight (FW).

2.4 Methanolic extraction

Fruit extracts were prepared as described by Qaderi et al. [20]. Firstly, 10 g of frozen fruit were homogenized with Ultraturrax T25 homogenizer (Janke and Kunkel, IKA Labortechnik, Staufen, Denmark) in 20 mL of methanol and agitated for 30 min in the dark. This solution was then centrifuged at 4500 rpm for 10 min at 4°C, the obtained supernatant was immediately stored at –20°C, while the pellet was extracted a second time by adding 20 mL of methanol and restarting the same process. After the suspension had been centrifuged at 4500 rpm for 10 min at 4°C, a second supernatant was obtained and added to the first one, then filtered (pore size 0.22μm) and finally stored at –20°C until injected into HPLC.

2.5 Vitamin C extraction

According to Tulipani et al. [21] description, fruits vitamin C was extracted using an ultrasound-assisted extraction procedure. The extraction was done using an ultrasonic bath (Bioblock/ELMA 88155, Stuttgart, Germany), a device that uses high-frequency electric current generated by a generator to create ultrasound waves inside a water tank. The method helps to accelerate the dissolving of solutes in some solvents.

The analysis requires homogenizing 1 g of frozen strawberry with an aliquot of 4 mL taken from the extraction buffer solution, containing 5% metaphosphoric acid and 1 mM DTPA, followed by 5 min of sonication and centrifugation at 4000 rpm for 10 min at 4°C. The supernatants obtained from each sample were filtered (filter pore size 0.45μm) and inserted into a vial to perform analysis on an HPLC system.

2.6 HPLC determination of Phenolic acids

The analysis of phenolic acids followed the procedures outlined in Schieber et al. [22] and Fredericks et al. [23]. The HPLC system was made up of a Jasco PU-2089 plus controller, a Jasco UV-2070 plus ultraviolet (UV) detector, and a Jasco AS-4050 autosampler (all from Jasco, Easton, MD, USA). The column utilized was an Aqua Luna C18 (250×4.6 mm) (Phenomenex, Torrance, CA, USA) protected by a Phenomenex 4.0×3.0 mm C18 ODS guard column (Phenomenex, Torrance, CA, USA). The HPLC UV detector was set at 320 nm. The gradient program consisted of two mobile phases: A (2% Acetic acid) and B (acetic acid, acetonitrile, and water 1 : 50 : 49). It started with 55% A and 45% B for 50 min, followed by 10 min 100% of B, and then decreased to 10 % B until the end.

For the quantification of phenolic acid content, external chlorogenic acid, caffeic acid, and ellagic acid calibration curves were used. Values were expressed as mg corresponding to phenolic acid per 100 g of fresh weight of strawberries (mg/100 g FW).

2.7 HPLC determination of Anthocyanins

Anthocyanin content was analyzed following the method of Fredericks et al. [23]. The HPLC system was made up of a Jasco PU-2089 plus controller, a Jasco UV-2070 plus ultraviolet (UV) detector, and a Jasco AS-4050 autosampler (all from Jasco, Easton, MD, USA). Monitoring was done at 520 nm, and the compounds were separated on an Aqua Luna C18 (250×4.6 mm) reverse-phase column with a particle size of 5μm (Phenomenex, Torrance, CA, USA) shielded by a Phenomenex 4.0×3.0 mm C18 ODS guard column. The gradient program consisted of two mobile phases: A (formic acid, acetonitrile, and water 10 : 3:87) and B (formic acid, acetonitrile, and H₂O 10 : 50 : 40). It started with 75% A for 10 min, decreased to 69% A for 5 min, decreased again to 60% A for 5 min, and later continued 50% A for 10 min, followed by 90% A for 16 min.

Anthocyanins were quantified using calibration curves made with external standards of cyanidin-3-glucoside, pelargonidin-3-glucoside, and pelargonidin-3-rutinoside, and were calculated as mg per 100 g of fresh weight of strawberries (mg/100 g FW).

2.8 HPLC determination of Vitamin C contents

Vitamin C content was measured as described by Helsper et al. [24]. Extracts were subjected to HPLC analysis after the extraction procedure. The UV (ultraviolet) detector Jasco UV-2070 plus (Jasco, Easton, MD, USA) was set at an absorbance of 260 nm, and the autosampler Jasco AS-4050 (Jasco, Easton, MD, USA) was part of the HPLC system. A Phenomenex 4.0×3.0 mm C18 ODS guard column (Phenomenex, Torrance, CA, USA) was utilized to protect the Ascentis Express C18 150×4.6 mm HPLC column (Supelco, Bellefonte, PA, USA). The gradient program consisted of two mobile phases: A (50 mM phosphate buffer with pH 3.2) and B (Acetonitrile), which started with 100% of A until 6 min, then decreased to 50% for 2 min, and again increased to 100% until the end.

The quantification of vitamin C content was carried out through a calibration curve prepared by running standard concentrations of vitamin C, and the results were expressed as mg vitC per 100 g fresh weight of strawberries (mg/100 g FW).

2.9 Data analyses

The results are presented as the mean values±standard error and were subjected to one-way analysis of variance (ANOVA), at a confidence level of 95%. Tukey’s tests were used to assess significant differences, and differences with a p-value of 0.05 or above were deemed significant. Statistical analyses were performed by using Statistica 7 software (StatSoft, TIBCO Software, Palo Alto, CA, USA).

3 Results and discussion

3.1 Inoculation and damage assessment from Botrytis cinerea

Fruits of the ‘Cristina’ cultivar were the most sensitive to Botrytis cinerea with almost 70% damage index, and ‘Romina’ fruits were the most tolerant in the control trial (35%) (Fig. 2). However, the interesting result, which was found in infected strawberries, shows that ‘Romina’ fruits are very susceptible (almost 100%) when infected with B. cinerea, and ‘Silvia’ fruits were the most tolerant having only 71.7% of infection development (Fig. 2). To better understand this result regarding ‘Romina’s behavior it must be necessary to conduct more analysis on other physical parameters. For example, in a similar experiment that we are conducting, we analyzed firmness data, and ‘Romina’ fruits resulted in the firmer among the analyzed genotypes (data not shown). This preliminary result can partially explain the behavior of ‘Romina’, which resulted as the most tolerant in the field (possibly for its high firmness) but became the most susceptible when manually infected.

Fig. 2

Susceptibility of strawberry cultivars to Botrytis cinerea (A: control trial; B: infected trial).

In general, our results agree with previous studies in literature, which also confirmed that the infection rate of strawberry fruits with B. cinerea is a characteristic that is associated with the genotype and, in some way, this characteristic can be considered intrinsic and genetically determined [13, 25]. In general, the infection with B. cinerea is effective in increasing postharvest decay of strawberry cultivars. Compared to the control, the infection significantly increases the McKinney’s Index and the severity decay index.

3.2 Total soluble solids content

The highest sugar content in control was detected in the fruits of the ‘Sibilla’ cultivar with an average of 7.9° Brix, and the lowest in the fruits of the ‘Silvia’ cultivar with an average of 4.82° Brix. ‘Romina’ and ‘Cristina’ fruits reach an average of 6.22 and 7.3° Brix respectively, and they show a slightly decreasing trend variation by the increasing of storage in all cultivars. The last changes were detected in ‘Silvia’ fruits, and the most decreasing trend was found in ‘Romina’ fruits (Fig. 3).

Fig. 3

Total Soluble solids content (°Brix) from day 0 to day 4 of ‘Romina’, ‘Cristina’, ‘Sibilla’, and ‘Silvia’ fruits. (A: control trials; B: infected trials). Different lowercase letters indicate significant differences for p≤0.05 for each cultivar on different days (Tukey test). Different uppercase letters indicate significant differences for p≤0.05 for different cultivars on the same day (Tukey test).

In infected fruit, the highest sugar content was achieved by the ‘Sibilla’ cultivar with an average of 7.78° Brix, and the lowest were the fruits of the ‘Silvia’ cultivar with an average of 5.22° Brix. ‘Romina’ and ‘Cristina’s fruits reach an average of 6.37 and 7.18° Brix respectively. From Fig. 3, it is possible to deduce how the ‘Cristina’ and ‘Silvia’ fruits show a significant decreasing trend variation till 4 days of storage, while, in the ‘Romina’ and ‘Sibilla’ cultivars, the treatment with Botrytis did not significantly affect the soluble solids content, recording a variation similar to untreated fruit (Fig. 3).

Based on the result, it is possible to identify a general decrease in the content of soluble solids in both infected and control fruits, from day 0 to day 4. The decrease is more marked in the fruits infected with Botrytis cinerea, in particular in the cultivars ‘Cristina’ and ‘Silvia’. In the ‘Cristina’ fruit, the value progressively decreases from 8.22° Brix on day 0 to 6.59° Brix on day 4 of treatment with Botrytis. In the ‘Silvia’ fruit, the value progressively decreases from 5.95° Brix on day 0 to 4.28° Brix on day 4 of treatment with Botrytis. In the control trial, both cultivars are rather stable over time, with a slight decrease in sugar content.

In the ‘Romina’ fruit, the decreasing trend was not very pronounced, only occurring after an initial increase observed between days 1 and 2. Soluble solids content progressively decreases from 5.88° Brix on day 0 to 5.14° Brix on day 4 of treatment with Botrytis, while in the control trial, the content decreases to 5.56° Brix on day 4.

In the ‘Sibilla’ fruit, the trend of the infected fruits records a significant progressive increase until day 3 and then decreases to 7.17° Brix on day 4. In the control fruit the trend is decreasing, and then remains stable and registers a slight increase to 7.76° Brix on day 4.

Unripe strawberries mostly include glucose and fructose, with small amounts of sucrose. Sucrose levels increase rapidly during de-greening and red coloring [26]. The higher increase of sugar content during fruit ripening can promote susceptibility as it can serve as a source of energy for promoting B. cinerea infection. Soluble sugars are the major carbon source for fungal growth and reproduction and may promote the infection processes [13]. Various research teams have endeavored to uncover the dynamic interaction between strawberry fruit and B. cinerea through transcriptome analysis. However, none of these investigations have offered insights into the involvement of sugars or sugar transporters in response to B. cinerea infection [27, 28]. In another study [13], a trend wherein a higher spoilage rate was associated with elevated levels of fructose and glucose was observed, but not with the level of sucrose or the overall sugar content. Despite sucrose being the primary factor contributing to variations in total sugar content, it appears that B. cinerea may not face a sugar deficiency upon infecting ripe fruit. Therefore, as also stated by Petrasch et al. [29], in the interaction between strawberry fruit and Botrytis, the abundance of sugars may not significantly contribute to fruit spoilage by grey mold.

3.3 Titratable acidity

In the control trial, the fruits of the ‘Sibilla’ cultivar had a higher citric acid content (0.77%), while the ‘Romina’ fruits were less acidic with an average value of 0.49%. The fruits of the ‘Silvia’ and ‘Cristina’ cultivars showed values of 0.66% and 0.72% respectively (Fig. 4). The evolution of the titratable acidity for the control trial of the cultivar ‘Cristina’ had an increasing trend after 4 days of storage, while, the ‘Romina’, ‘Sibilla’, and ‘Silvia’ control trials showed a significant decrease. The evolution of titratable acidity for the fruit infected with Botrytis cinerea varied depending on the cultivar (Fig. 4): ‘Romina’, ‘Cristina’, and ‘Sibilla’ had an inverse variation compared to that observed for sugars from day 0 to day 4, registering an increase of fruit titratable acidity, while the ‘Silvia’ cultivar recorded decreases in values.

Fig. 4

Titratable acidity (% citric acid) from day 0 to day 4 of ‘Romina’, ‘Cristina’, ‘Sibilla’, and ‘Silvia’ fruits. (A: control trials; B: infected trials). Different lowercase letters indicate significant differences for p≤0.05 for each cultivar on different days (Tukey test). Different uppercase letters indicate significant differences for p≤0.05 for different cultivars on the same day (Tukey test).

In ‘Romina’ fruit, the value progressively increases from 0.46% on day 0 to 0.66% on day 3 of treatment with Botrytis and then decreases on day 4 to 0.48%. A different trend is followed in the control trial, in which the value progressively decreases until day 4.

In ‘Cristina’ fruit, the value decreases from 0.74% on day 0 to 0.68% on day 3 of treatment with Botrytis and then increases on day 4 to 0.89%. A similar trend is followed in the control trial, in which the value progressively decreases until day 3, and then increases on day 4 to 0.76%.

In ‘Sibilla’ fruit, the value decreases from 0.75% on day 0 to 0.63% on day 2 of treatment with Botrytis and then increases on day 4 to 0.93%. A similar trend is followed in the control trial, in which the value progressively decreases until day 3, and then increases on day 4 to 0.72%.

In ‘Silvia’ fruit, the total acidity initially decreases on day 1, and then gradually increases up to day 4 at 0.68%, after treatment with Botrytis. A different trend is followed in the control test, in which the value progressively decreases from 0.75% on day 0 to 0.54% on day 3. B. cinerea can alter neutral sugar and sugar acid levels in the infected host tissues, mainly by degradation and depolymerization of cell walls [30].

3.4 Phenolic acids

Phenolic acids are important secondary plant metabolites that have attracted considerable interest in the past few years due to their many potential health benefits, as they are powerful antioxidants. Therefore, it is important to verify their presence.

In the control trial, the highest phenolic acid content was detected in ‘Silvia’ fruits (25.67 mg/100 g FW), while the lowest amount was achieved in ‘Cristina’ fruits with 16.64 mg/100 g FW. ‘Romina’ and ‘Sibilla’ fruits contained 17.9 mg/100 g FW and 22.75 mg/100 g FW at day 0, respectively (Fig. 5).

Fig. 5

Phenolic acids content of fruits from day 0 to day 4 of ‘Cristina’, ‘Romina’, ‘Sibilla’, and ‘Silvia’ fruits. (A: control trials; B: infected trials). Different lowercase letters indicate significant differences for p≤0.05 for each cultivar on different days (Tukey test). Different uppercase letters indicate significant differences for p≤0.05 for different cultivars on the same day (Tukey test).

All cultivars in control fruits had increased phenolic acids from day 0 till day 4, except for ‘Cristina’ in day 2 (6.55 mg/100 g FW). In detail, ‘Cristina’ fruits had significantly increased phenolic acids from 16.64 mg/100 g FW to 27.03 mg/100 g FW on day 4, with a decrease on day 2, and in ‘Romina’, ‘Sibilla’, and ‘Silvia’ phenolic acids progressively increased from 17.9 mg/100 g FW, 22.75 mg/100 g FW, and 25.67 mg/100 g FW at day 0, to 22.68 mg/100 g FW, 34.42 mg/100 g FW and 33.80 mg/100 g FW at day 3, then decreased to 21.97 mg/100 g FW, 29.19 mg/100 g FW and 30.37 mg/100 g FW, respectively.

Regarding infected fruits, the highest phenolic acid content was detected in ‘Silvia’ (29.99 mg/100 g FW) and the lowest in ‘Romina’ fruits (16.31 mg/100 g FW), and phenolic acid content of ‘Cristina’ and ‘Sibilla’ fruits was 18.25 mg/100 g FW and 23.58 mg/100 g FW, respectively at day 0 (Fig. 5).

Generally, in all cultivars, there was an increasing trend in phenolic acids fruit content from day 0 to day 3, and at day 4 phenolic acids decreased in all cultivars. In ‘Cristina’, the phenolic acids increased from day 0 to day 4 except on day 2, in which was observed the lowest content during storage (9.91 mg/100 g FW), similar to the control trial. In ‘Romina’, ‘Sibilla’, and ‘Silvia’ fruits phenolic acids increased from day 0 to day 2, while from day 2 to day 4 had decreased trend.

As a comparison, infected fruits had more phenolic acid content compared to control at the middle storage period but high phenolic acid content was achieved in control at the end of day 4 compared to infected fruits.

Strawberry fruits are a good source of natural antioxidants whose extracts exhibit high enzymatic activity against free radicals and oxygen detoxification [31]. Numerous studies have reported that the antioxidant properties of constitutive and induced phenolic compounds provide good defense for plant tissue against pathogen intrusions [32, 33].

In strawberry cultivars, the main phenolic components having antioxidant properties are ellagic and gallic acids, which confer a higher tolerance to B. cinerea. In a study by Hegyi Kalo et al. [25] was observed that the inhibition of Botrytis cinerea growth was more profound on dark berries than on white berries that contain lower concentrations of phenolic compounds. In another study, Harshman et al. [34] reported that total phenolics have the greatest positive correlation with tolerance to decay: as phenolics increase, tolerance to decay also increases, but contrariwise in this study, no correlation was obtained (Fig. 6).

Fig. 6

The distribution between fruit phenolic acids content and Decay severity.

In summary, the phenolic acids content of strawberry fruit may change due mainly to their genetics but also to their morphology and anatomy, which could also influence their susceptibility to B. cinerea. Further investigation is then required for a better understanding.

3.5 Anthocyanins

Not infected fruit of ‘Silvia’ had the highest anthocyanin content (45.73 mg/100 g FW), while it was the lowest in ‘Cristina’ fruits (27.56 mg/100 g FW); in ‘Sibilla’ and ‘Romina’ fruits, the amount of anthocyanin content was also detected and resulted in 34.45 mg/100 g FW and 40.6 mg/100 g FW, respectively, at day 0 (Fig. 7). Fruit of ‘Cristina’, ‘Sibilla’, and ‘Silvia’ showed an increased trend in fruit anthocyanins content from day 1 to day 3, followed by a decrease on day 4, while ‘Romina’ fruit had a strong increasing trend from day 1 to day 4. ‘Romina’ fruit accumulated the highest amount of anthocyanin, up to 153.68 mg/100 g FW on day 4.

Fig. 7

Total anthocyanin content of fruits from day 0 to day 4 of ‘Cristina’, ‘Romina’, ‘Sibilla’, and ‘Silvia’ fruits. (A: control trials; B: infected trials). Different lowercase letters indicate significant differences for p≤0.05 for each cultivar on different days (Tukey test). Different uppercase letters indicate significant differences for p≤0.05 for different cultivars on the same day (Tukey test).

Regarding infected fruits, ‘Silvia’ cultivars had the highest amount of anthocyanin content with 51.01 mg/100 g FW and ‘Cristina’ the lowest with 29.11 mg/100 g FW at day 0, while in ‘Romina’ and ‘Sibilla’ fruits were detected at 45.81 mg/100 g FW and 49.17 mg/100 g FW, respectively (Fig. 7). However, no significant difference was detected among cultivars.

In infected fruits, there is no clear anthocyanin content trend in any cultivar during the 4 days of storage. Each cultivar seems to respond differently. In detail, ‘Cristina’ had slightly increased from day 0 to day 2 and then decreased till day 4 with the amount of 17.37 mg/100 g FW. ‘Romina’ fruit had a high accumulation on day 1 (113.64 mg/100 g FW), followed by a strong decrease on day 4 (3.4 mg/100 g FW). In ‘Sibilla’ fruit, the anthocyanin content reached a maximum peak on day 0 of 49.17 mg/100 g FW, then from day 1 till day 4 had decreased trend (11 mg/100 g FW); in ‘Silvia’ fruit, anthocyanin content increased at day 1 (72.54 mg/100 g FW) and decreased from day 2 till day 4.

As a comparison to the control trial, infected fruits for all cultivars had much less amount of anthocyanin content. In control fruits, anthocyanin content was increased because of increasing fruit ripening during post-harvest, while in infected fruit anthocyanins decreased due to enzymatic activity degradation. A high amount of anthocyanin content was demonstrated to reduce the susceptibility to Botrytis due to their antioxidant activity [30]. Our results agree with this finding, as strawberry cultivars displaying lower fruit spoilage rates had more anthocyanins. In addition, the distribution between anthocyanin and Botrytis shows that there is a positive correlation: as anthocyanins increase, tolerance to decay also increases (Fig. 8). Enhancing anthocyanin content through genetic modification has been shown to decrease susceptibility to B. cinerea in tomatoes, likely due to its antioxidant properties [35]. In another study [13], has been shown that strawberry genotypes with lower spoilage rates exhibited a more vibrant red color, which showed a positive correlation with the overall anthocyanin levels. However, there was no significant correlation between the total anthocyanin levels and the spoilage rate. The different relations between strawberry red color, anthocyanin content, and susceptibility to B. cinerea may be attributed to the observation that the external and internal coloration, as well as anthocyanin accumulation in strawberries, are not necessarily synchronized [13].

Fig. 8

The distribution between total anthocyanin content and Decay severity.

3.6 Vitamin C content

In control fruits, the highest content of vitamin C was detected in ‘Sibilla’ (36.29 mg/100 g FW), and the lowest amount was achieved in ‘Cristina’ (13.41 mg/100 g FW), while ‘Romina’ and ‘Silvia’ had 28.44 mg/100 g FW and 30.53 mg/100 g FW, at day 0, respectively (Fig. 9).

Fig. 9

The vitamin C content of fruits from day 0 to day 4 of ‘Cristina’, ‘Romina’, ‘Sibilla’, and ‘Silvia’ fruits. (A: control trials; B: infected trials). Different lowercase letters indicate significant differences for p≤0.05 for each cultivar on different days (Tukey test). Different uppercase letters indicate significant differences for p≤0.05 for different cultivars on the same day (Tukey test).

Generally, fruits and vegetables show a gradual decrease in vitamin C content as the storage temperature or duration increases [36]. In this study, in untreated fruits (control trial), for the 4 cultivars, there is no clear or general vitamin C content trend during 4 days of storage. Each cultivar seems to respond differently: in the ‘Romina’ fruit, the vitamin C content reaches a maximum peak on day 1. The value progressively decreases to 26.1 mg/100 g FW on day 3, recording a slight increase on day 4.

In ‘Cristina’ fruit, the vitamin C content increases progressively, reaching a maximum peak on day 2 of 19.21 mg/100 g FW, and then decreases until day 4.

In ‘Sibilla’ fruit, the vitamin C content reaches a maximum peak on day 0 of 36.3 mg/100 g FW, then from day 1 till day 4 had an almost constant value.

In ‘Silvia’ fruit, the vitamin C content shows an oscillating trend, consisting of decreases and increases that follow one another from day 0 to day 4, recording a maximum peak on day 0 of 30.53 mg/100 g FW.

In infected fruit, the highest vitamin C content was detected in the ‘Sibilla’ fruit, with an average of 36.2 mg/100 g FW, and the lowest in the fruits of the ‘Silvia’ cultivar with an average of 12.18 mg/100 g FW. ‘Romina’ and ‘Cristina’ fruit reached an average of 27.7 and 17.57 mg/100 g FW respectively, at day 0. ‘Cristina’, ‘Sibilla’, and ‘Silvia’ fruits showed a marked decreasing trend after 4 days of storage, while ‘Romina’ had increased at day 1, then progressively decreased till day 4 (Fig. 9). The treatment with B. cinerea significantly affected the vitamin C content, since at the end of day 4 all cultivars had lost almost all the vitamin C. Control fruit had much more vitamin C content compared to infected fruits.

During the B. cinerea infection, Bui et al. [37] noticed a rise in the enzymatic activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX), along with a drop in the ascorbic acid content in fruit. They suggested that as SOD is crucial for detoxifying reactive oxygen species generated by either fruit or fungi, the increase in SOD activity may have arisen from fruit or B. cinerea in response to oxidative stress [38]. Ascorbic acid is no longer recycled but consumed by APX in fruits with increasing severity of Botrytis cinerea infection, due to disruption in redox equilibrium. Similar findings were also detected in the present study, with the vitamin C content that showed a negative linear correlation with the increasing severity of fruit decay (Fig. 10).

Fig. 10

The distribution between fruit Vitamin C content and Decay severity.

4 Conclusions

For the present study, four well-defined strawberry cultivars (‘Romina’, ‘Cristina’, ‘Silvia’, and ‘Sibilla’) were infected with B. cinerea, the fungus that causes grey mold. The susceptibility response of each cultivar to the disease caused by the fungal pathogen was evaluated in comparison with the analyses of fruit content of fruit qualitative and nutritional compounds, to identify the possible correlation between higher tolerance to B. cinerea infection and fruit accumulation of compounds related to fruit quality and nutritional value.

The contamination with B. cinerea is effective in increasing the postharvest decay of strawberry cultivars after 4 days of storage. McKinney’s index of strawberry fruits was significantly increased by Botrytis treatment for all cultivars. Among the strawberry cultivars analyzed, the ‘Silvia’ cultivar is the one that demonstrated a higher level of tolerance to Botrytis infection during the treatment trial, compared to the ‘Romina’, ‘Cristina’, and ‘Sibilla’ cultivars. ‘Romina’ cultivar on day 4 was assessed at grade 5 (81% to 100% fruit surface infected and showing sporulation), resulting in the most susceptible cultivar to the treatment. It is possible to identify a general decrease in the content of soluble solids in both infected and control fruits, from day 0 to day 4. However, on day 4, compared to the control trial, the cultivars ‘Romina’, ‘Cristina’, ‘Silvia’, and ‘Sibilla’ contaminated with B. cinerea recorded a decrease in the soluble solids content. The contamination with B. cinerea is effective also in increasing the content of free acids of strawberry cultivars after 4 days of storage, compared to the control trial. The evolution of titratable acidity for the fruit infected with Botrytis cinerea varied depending on the cultivar. On day 4, compared to the control trial, it is possible to identify an increase in the free acid content for the ‘Sibilla’, ‘Silvia’, ‘Cristina’, and ‘Romina’ cultivars in the infected trial. The results of the study also highlighted important nutritional changes in the four strawberry cultivars contaminated with B. cinerea.

Generally, in all cultivars, phenolic acids, and vitamin C decreased in both control and infected fruits, but there was a strong decrease in infected fruit.

Anthocyanin content increased in control fruits but strongly decreased in infected fruits. A strong positive correlation was obtained between fruit nutritional quality and tolerance to decay, as a high amount of nutritional content resulted in a higher tolerance to Botrytis cinerea. However, it is important to consider that aspects other than nutritional quality can influence the capacity of strawberry fruits to tolerate B. cinerea infection, such as physical properties and fruit senescence after detachment from the plant. Therefore, to better understand the mechanism of tolerance of strawberry fruits to infections from pathogens, other aspects should be considered in future research.

Acknowledgments

The authors have no acknowledgments.

Funding

This research was funded by the Partnership for Research and Innovation in the Mediterranean Area (PRIMA)’s 2019–2022 MEDBERRY project.

Conflict of interest

Luca Mazzoni and Bruno Mezzetti are Editorial Board Members of this journal but were not involved in the peer-review process nor had access to any information regarding its peer-review.

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