Degree of deterioration of the chemical composition of apricot fruits caused by Sharka disease

This trial was carried out in private commercial plantation located in Prislonica vilage near Čačak city, western Serbia in 2023 and 2024. Main weather conditions during investigation and long-term average values (monthly air temperature in °C and sum of precipitation in mm) are presented in Tab. 1. Study area is located at 340 m above sea level, 43°53’ N latitude, 20°21’ E longitude. Orchard was established in 2015 with 19 apricot cultivars originating from different world regions, including ‘Precoce de Thyrinte’ as plant material. Before grafting, scion shoots of all cultivars were tested on all known PPV strains using IC-RT-PCR procedure and declared as virus-free. After this procedure, scion genotype was grafted onto Myrobalan seedlings in summer 2014 and planted in autumn 2015 with spacing of 5.5 m × 3.0 m. Standard cultural practices were used, except irrigation.

The trial was set up in a randomized block design with four replications, each containing five trees of the tested ‘Precoce de Thyrinte’ cultivar (n = 20).

As a result of natural infection, the first visible symptoms of PPV presence on this cultivar were observed on fruits and stones in 2020 and 2021 on six trees in total. The infected trees were marked and subjected to observation and analysis and compared with 6 PPV uninfected trees in 2023 and 2024. Leaf samples (average 20 per tree) for presence of PPV were taken randomly in mid-May from all parts of the canopy from 6 trees with visible PPV symptoms. By random selection, 6 trees with no visible PPV symptoms were marked and 20 leaves per tree were collected at the same time.

PPV detection and characterization was carried out with Immunocapture Reverse Transcription Polymerase Chain Reaction (IC-RT-PCR) in May 15, 2020 on apricot leaves. Polyclonal antibodies (0.5 mg mL^‒1) produced at the Fruit Research Institute, Čačak were used for PCR plate coating in immunocapture step. Reverse transcription was performed with random hexamer primers using Maxima Reverse Transcriptase (Thermo Fisher Scientific, USA). The obtained cDNA was used for further PCR analyses. Each sample was tested in three separate PCR reactions using primers designed by Šubr et al. (2004). Primer sets mD5/mD3, mM5/mM3, and mD5/mM3, that amplify the 3’NIb-5’CP region, were used for specific detection of PPV-D, PPV-M and PPV-Rec isolates, respectively. PCR mixture of 20 µl consisted of 2µl10 × DreamTaq Green Buffer, 2 µl 2.5 mM dNTPs, 1.2 µl 25 mM MgCl₂, 0.2 µl of each 10 mM primer, and 0.2 µl 5U of Green Taq DNA polymerase (Thermo Fisher Scientific, USA). The thermal cycling conditions were as follows: 3 min at 94°C; 30 cycles each of 45 s at 94°C, 30 s at 60°C, and 60 s at 72°C; and a final step of 7 min at 72°C. All PCRs were carried out in TPersonal thermal cycler (Biometra, Germany). Obtained PCR products were analyzed in 1.5% agarose gel, stained with ethidium-bromide, and visualized in a Gel Doc EZ System (Biorad, USA). The presence of the PCR fragment of the expected size was considered a positive reaction.

For each sampling, 10 PPV-uninfected fruits were randomly hand-picked from trees showing no visible PPV symptoms (n = 40), and 10 symptomatic fruits (Fig. 1) were collected from PPV-infected trees. Fruits were sampled from all parts of the canopy at full ripening stage (approximately mid-June in both years), with four replications per group (n = 40).

Fig. 1:

Typical symptomatic fruits on a PPV-M-infected apricot cultivar ‘Precoce de Thyrinte’ tree in Prislonica village near Čačak city, Serbia (photo taken in mid-June 2024 by T. Milošević).

Fruit weight (FW, g) was measured using digital balance VK-TECH (Daejeon, South Korea). The juice for chemical analyses was extracted using kitchen extractor from 10 randomly selected whole fruits (skin + pulp) from PPV uninfected and PPV infected trees. The SSC (°Brix) was assessed with a hand refractometer CZ 32-G (Carl Zeiss, Jena, Germany) at 20 °C. For both malic (%) and citric acid (%), prepared juice was titrated with 0.1 mol L^‒1 NaOH, up to pH 8.1 using 1–2 drops of phenolphthalein as an indicator to pink. Once the SSC and malic acid content were assessed, the ripening index (RI) or SSC/malic acid ratio was calculated. Juice pH was measured with a pH meter MA 5740 (Iskra, Kranj, Slovenia). For ash content (%) determination, fruits were incinerated at 550°C in oven during 8–10 h. Sugar contents (TS, RS, IS and SU (all in % of fresh weight, fw)] were determined according to the Luff-Schoorl method (AOAC, 1995). The SI was expressed as TS/malic acid ratio.

Spectrophotometric measurements for chlorophyll (a and b), total carotenoids, TPC, TFC and TAC were performed by Cary 300 UV/Vis spectrophotometer (Agilent Technologies, Santa Clara, CA) on fw basis. Contents of chlorophyll (a and b) and total carotenoids were determined by methods proposed by Lichtenthaler and Wellburn (1983), and the results were expressed as μg 100 g^‒1. The TPC (mg 100 g^‒1) was determined using the Folin-Ciocalteu method (Singleton et al., 1999) whereas TFC (mg 100 g^‒1) was estimated by the AlCl₃ method (Quettier et al., 2000). The TAC (mg 100 g^‒1) was evaluated by the phosphor-molybdenum method (Prieto et al., 1999).

The values presented for each measurement are the means of triplicate measures on equidistant points of each fruit and expressed as the mean ± standard error (SE).

The results were processed statistically by two-way analysis of variance (ANOVA), model 2 × 2, using MS-Excel software (Microsoft Corporation, Redmond, WA, USA). Source of variations was fruit viral status (A) and year (B). When the F test was significant, means were compared with the LSD test at P ≤ 0.05.

Results of the IC-RT-PCR analysis revealed that only PPV-M strain was detected in analyzed samples. PPV was confirmed in all samples showing Sharka-like symptoms on fruits and leaves. In asymptomatic samples, the PPV presence was not confirmed. The PPV symptoms on apricot fruits in our study are given in Fig. 1. PPV-M strain is present in many European countries. It is considered an epidemic strain that is efficiently transmitted by a number of aphid species (Dallot et al., 2003; Capote et al., 2010). Otherwise, susceptible apricot cultivars were characterised by high relative PPV concentration (9.8 × 10^‒5) whereas cultivars resistant to PPV were characterised by very low relative PPV concentration (5 × 10^‒2) (Polák, 1998).

Data in Tab. 2 showed that FW from PPV symptomatic trees was lower by 22.64% on average compared to PPV asymptomatic trees. Several authors reported that diseased apricot fruits are smaller and deformed (Llacer and Cambra, 1986; Poggi Pollini et al., 2008) with deformations accompanied by typical rings and mottling. On the other hand, FW was higher in first season of trial than in second. Interaction viral status × year was not significant. Other authors emphasize the significant influence of climatic factors (air temperature, amount of precipitation) on the spread of PPV and intensity of infection (Špánik et al., 1998; Glasa et al., 2003). In our earlier study, we found that there were no significant differences in FW of ‘Hungarian Best’ (clone ‘T-14’) harvested from PPV infected and healthy trees, although a mixed infection with PPV-D and PPV-Rec strains was detected in this apricot (Milošević et al., 2019).

Healthy fruits had higher SSC and better RI value than diseased ones, whereas diseased fruits had higher values of both malic and citric acids and ash content (Tab. 3). There were no significant differences between PPV uninfected and PPV infected fruits in pH juice. All sugar contents and SI were higher in fruits picked from uninfected trees (Tab. 4). For example, healthy fruits contained 13.67% more TS than PPV infected ones. These data are in agreement with results from Usenik et al. (2015) and Espinoza et al. (2021) who reported that PPV infection can modify the ripening process, induced by an alteration of the primary metabolism, including organic acids, soluble solids and sugars. Similarly, previous studies recorded that PPV infected fruits were lower in sugars (Šutić et al., 1999; Usenik and Virscek Marn, 2017), especially in sucrose (Kamenova and Milusheva, 2005) and higher in acidity, resulting in inferior taste of diseased fruits (Milošević et al., 2019). On the other hand, Poggi Pollini et al. (2008) reported that fruit quality in general was only negligibly influenced by the infection but only SSC appeared to be slightly higher in fruits from PPV unifected trees compared to the symptomatic fruits of the same genotype. Probably, intensity and duration of infection, fruit type, genotype, crop load, nutrient status of the plant and tree age may be the reason for the discrepancies between the results of the mentioned authors as previously reported by Vanek et al. (1992).

Comparing the results by years of research, statistically higher SSC, pH juice, ash content, IS, SU and both ripening and sweetness indexes were observed in 2023, whereas amounts of both malic and citric acids, RS and TS were higher in 2024. These results can be connected to the influence of weather conditions (Vanek et al., 1992), especially temperature on pathogenicity (aggressivity) of PPV (Glasa et al., 2003).

Data in Tab. 5 and 6 showed that content of pigments and secondary metabolites evaluated were higher in fruits picked from uninfected trees than in infected fruits. For example, there were 1.56- and 1.21-fold differences between higher and lower values of total chlorophyll and total carotenoid contents, respectively i.e. between healthy and diseased fruits. According to Hernández et al. (2006), PPV infections produce alterations in the chlorophyll fluorescence parameters in susceptible apricot cultivars such as ‘Real Fino’ which was confirmed by our results. In addition, the alteration of the photosynthesis provoked by a PPV infection could affect the primary metabolism in the plant (Clemente-Moreno et al., 2014).

As known, TPC and TFC are important as they provide information on the overall quality of apricot fruits, especially as human health-promoting compounds. Previous research has shown that Sharka disease significantly modifies the above mentioned phytochemicals in sensitive stone fruits, especially plum (Usenik et al., 2015). This may be associated with the enhanced synthesis and accumulation of these compounds, especially flavonoids, in order to defend plants against stresses of biotic and abiotic origin. In light of this, Horsáková et al. (2013) reported that increased content of antioxidant activity in peach fruits of PPV infected trees is probably caused by the function of protective systems which regulate the production of reactive oxygen species and thus protect cells from oxidative damage. However, the results in our work showed the opposite trend, i.e. healthy fruits were richer in TPC and TFC than diseased ones. In our previous study on ‘Hungarian Best’ apricot (Milošević et al., 2019), we also determined significantly higher TPC and TFC content in healthy fruits compared to PPV infected ones. Interestingly, total antioxidant activity in our study was equal in both uninfected and infected fruits (Tab. 6). It seems that fruit type, cultivar and PPV strain play important roles in the degree of the plant’s reaction to its presence.

Significant differences were observed in the effects of the year on pigments and phenolic compounds content and antioxidant activity (Tab. 5 and 6). The chlorophyll a (Chl a), TPC, TFC and TAC were higher in 2023 than in 2024, whereas chlorophyll b (Chl b) and total carotenoids were higher in 2024. Total chlorophyll content was similar in both years of investigation. Year-to-year variations in pigments and secondary metabolites content in PPV infected fruits such as peach and/or plums have been well documented in previous studies (Samara et al., 2017; Miletic et al., 2022). In addition, Miletic et al. (2022) reported that plum fruit infected with PPV was richer in some phytochemicals under heavy rainfall during summer months. In the present study, a very high amount of precipitation during May and June i.e. in the last stage of fruit development and ripening was registered (Tab. 1) and such weather conditions could have affected the content of these compounds.

However, the significant interaction viral status × year demonstrates the complex nature of the trends in the sources of variation for all primary and secondary metabolites (Tab. 5 and 6). Namely, the tendency that viral status was a key category in determining the content of primary and secondary metabolites over the years is not consistent. Contrary to our results, Miletic et al. (2022) reported that interactions viral status × year were not significant for TPC and TFC in plum cv. ‘Čačanska Lepotica’. These discrepancies may be related to the fruit type, cultivar, PPV strain and its virulence capacity, duration of infection, tree age, weather conditions and orchard management as previously reported (Vanek et al., 1992; Glasa et al., 2003; Usenik et al., 2015; Milošević et al., 2019). For example, mineral nutrition can reduce or enhance the intensity of PPV symptoms (Nemeth, 1986).