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10 April 2023

Effect of Rootstock on the Volatile Profile of Mandarins

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1
Instituto Valenciano de Investigaciones Agrarias (IVIA), 46113 Moncada, Spain
2
Centro de Investigación e Innovación Agroalimentaria y Agroambiental (CIAGRO-UMH), Ctra. Beniel, km 3.2, 03312 Orihuela, Spain
3
Laboratorio de Fitoquímica y Alimentos saludables (LabFAS), Departmento de Ciencia y Tecnología de Alimentos, Centro de Edafología y Biología Aplicada del Segura (CEBAS)-CSIC, University Campus-25, 30100 Murcia, Spain
4
Postharvest and Refrigeration Group, Department of Agronomical Engineering and Institute of Plant Bio-Technology, Universidad Politécnica de Cartagena, 30203 Murcia, Spain
This article belongs to the Special Issue Fruits and Fruit-Based Products as a Source of Bioactive Compounds

Abstract

Mandarin production has increased in recent years, especially for fresh consumption, due to its ease of peeling, its aroma, and its content of bioactive compounds. In this sense, aromas play a fundamental role in the sensory quality of this fruit. The selection of the appropriate rootstock is crucial for the success of the crop and its quality. Therefore, the objective of this study was to identify the influence of 9 rootstocks (“Carrizo citrange”, “Swingle citrumelo CPB 4475”, “Macrophylla”, “Volkameriana”, “Forner-Alcaide 5”, “Forner-Alcaide V17”, “C-35”, “Forner-Alcaide 418”, and “Forner-Alcaide 517”) on the volatile composition of “Clemenules” mandarin. For this, the volatile compounds of mandarin juice were measured using headspace solid-phase micro-extraction in a gas chromatograph coupled to a mass spectrometer (GC-MS). Seventy-one volatile compounds were identified in the analyzed samples, with limonene being the main compound. The results obtained showed that the rootstock used in the cultivation of mandarins affects the volatile content of the juice, with “Carrizo citrange”, “Forner-Alcaide 5”, “Forner-Alcaide 418”, and “Forner-Alcaide 517” being those that presented the highest concentration.

1. Introduction

Citrus is one of the main cultivated fruits worldwide [1]. Among the different citrus fruits (oranges, lemons, limes, grapefruit, and mandarins), the mandarin (Citrus reticulata) is gaining popularity due to its economic and nutritional value [2]. Mandarin production has reached 38 million tons in 2020 [3]. Currently, China is the largest producer of mandarins (23.12 mln. tons), followed by Spain (2.17 mln. tons), Turkey (1.58 mln. tons), and Brazil (1.02 mln. tons) [3]. Throughout the world, citrus fruits are one of the most important fruits, especially in juice production [4,5]. However, mandarins are mainly consumed fresh, although they have a shorter shelf life than other citrus fruits [6]. In this sense, Spain has had notable success with its seedless clementine varieties in Europe and the United States [7]. The main reasons for the fresh consumption of mandarins are that they are easy to peel; have a desirable flavor; and their content of vitamin C (≈25.8 mg/100 mL), flavonoids (≈38.97 mg rutin equivalent g−1 DW), and total phenolics (≈59.3 mg GAE/100 mL) [6,8]. The presence of citrus phenolic compounds contributes to the sensory quality of the fruit, in addition to being associated with the reduction of cardiovascular diseases and some types of cancer [9,10]. Moreover, aromas and volatile compounds play a fundamental role since they are responsible for the flavor of the fruit, so aromas are an important contributor to the sensory quality of these fruits and their derivatives [11,12,13].
On the other hand, farmers depend not only on the yield but also on the quality of the fruit [14]. In this sense, rootstocks play an important role since they help crops adapt to climate and soil conditions, as well as being a method of defense against climate change [9]. The selection of the appropriate rootstock is crucial for the success of the crop [15]. The identification of markers linked to citrus flavor and aroma can facilitate genetic improvement and the release of new superior varieties [8]. Some authors have shown that rootstocks affect the quality of citrus fruits, for example soluble solids content, acidity, ripening index, composition sugars and organic acids, antioxidant activity, and total phenolics, among others [14,16,17]. Currently, consumers demand higher-quality fruit that is produced sustainably [18]. Therefore, obtaining higher-quality citrus (internal and external) is essential. Then, the new studies carried out no longer focus exclusively on the yield and optimization of crops but instead choose to evaluate the effect of rootstocks on the quality of fruits [9]. Furthermore, there is little information on the effect of rootstock on volatile compounds in citrus.
For all the above-mentioned reasons, the objective of this study was to identify the influence of 9 generative rootstocks (“Carrizo citrange”, “Swingle citrumelo CPB 4475”, “Macrophylla”, “Volkameriana”, “Forner-Alcaide 5”, “Forner-Alcaide V17”, “C-35”, “Forner-Alcaide 418”, and “Forner-Alcaide 517”) on the volatile composition of “Clemenules” mandarin (Citrus clementina Hort. ex Tan.). This information can be used to improve the citrus market, which can provide sustainable economic opportunities for growers and be useful in promoting the use of rootstocks that induce greater citrus aroma.

2. Materials and Methods

2.1. Plant Material

“Carrizo citrange”, “Swingle citrumelo CPB 4475”, “Macrophylla”, “Volkameriana”, “C-35”, and four new hybrid selections, obtained in the rootstock breeding program carried out at IVIA (Instituto Valenciano de Investigaciones Agrarias) since 1974 (Table 1), were tested as rootstocks for “Clemenules” (selection virus-free INIASEL 22). Seeds of “Carrizo” citrange and “Cleopatra” mandarin were obtained from the germplasm collection of rootstocks at IVIA, and the seeds of the hybrids were obtained from the plants obtained in the citrus rootstock breeding program.
Table 1. Pedigree of the nine rootstocks tested for “Clemenules” mandarin.
The trial was located in Museros, at ANECOOP’s “Masía del Doctor” (Valencia, Spain). The soil type of the trial plot as well as the fertilization applied were those described by Legua et al. [17].

2.2. Preparation of Juice

The mandarin “Clemenules” (Citrus clementina Hort. ex Tan.) fruits were harvested at optimum maturity (>12 °Brix). The juice preparation was carried out according to the methodology proposed by Legua et al. [17].

2.3. Volatile Composition

The determination of volatile compounds in the mandarin juice was carried out following the method described by Cano-Lamadrid et al. [19], using the headspace solid-phase micro-extraction (HS-SPME) method with slight modifications. A SPME 50/30 mm DVB/CAR/PDMS (Divinylbenzene/Carboxen/Polydimethylsiloxane) fiber (Supelco) was used for the extraction. The exposure time was 50 min at a temperature of 40 °C and with constant agitation (600 rpm). Then, desorption of the volatile compounds from the fiber was carried out in the injection port of the gas chromatograph for 3 min at 230 °C. Volatile compounds were analyzed and identified using a Shimadzu GC-17A gas chromatograph coupled to a Shimadzu QP-5050A mass spectrometer (Shimadzu Corporation, Kyoto, Japan). The analysis was carried out from 45 to 400 m/z with an electronic impact (EI) of 70 eV in 1 scan/s mode. The GC-MS system consisted of a TRACSIL Meta X5 column containing 95% dimethylpolysiloxane and 5% diphenylpolysiloxane (Teknokroma S. Co., Ltd., Barcelona, Spain; 30 m × 0.25 mm i.d., 0.25 µm film thickness). The oven program started at 80 °C with an increase of 3 °C/min from 80 °C to 210 °C and hold for 1 min. After this, an increase of 25 °C/min from 210 °C to 300 °C was maintained for 3 min. The injector and detector temperatures were 230 and 300 °C, respectively. Helium was used as the carrier gas (column flow rate of 0.6 mL/min).
Three methods were used to identify volatile compounds: (i) retention rates and their comparison with the literature; (ii) retention times of pure chemical compounds; (iii) mass spectra of authentic chemical compounds and the spectral library of the National Institute of Standards and Technology (NIST) database. Only fully identified compounds have been described. The analysis of the volatile composition was run in triplicate.

2.4. Statistical Analysis

To carry out the statistical analysis, the software XLSTAT (Addinsoft 2016.02.270444 version, Paris, France) was used. Two-way analysis of variance (ANOVA) and Tukey’s multiple range test were used to compare experimental data and determine significant differences between rootstocks (p < 0.05). Principal component analysis (PCA) using Pearson correlation was also run.

3. Results and Discussion

A total of 71 volatile compounds (Table 2) were identified in the analyzed samples. Limonene stands out among the 10 main compounds (Table 3), with an average of 7998.4 µg L−1, which was expected since it is the main volatile compound in citrus [20,21], followed by: myrcene (293.7 µg L−1), linalool (247.4 µg L−1), valencene (122.1 µg L−1), decanal (119.9 µg L−1), ethanol (106.4 µg L−1), ethyl butyrate (84.8 µg L−1), terpinen-4-ol (80.5 µg L−1), octanal (65.5 µg L−1), and 1-octanol (40.7 µg L−1). It is interesting to note that limonene and valencene may affect the perception of other volatiles [22,23].
Table 2. Retention indexes (RT), kovats indexes (KI EXP: kovats index experimental, and LIT: kovats index literature), and principal descriptors of the volatile compounds identified in “Clemenules” mandarin juice [16,24,25].
Table 3. Concentrations (µg L−1) of volatile compounds in “Clemenules” mandarin (Citrus clementina Hort. ex Tan.) juice.
Looking at the main compounds detected, the “Forner-Alcaide 517” rootstock obtained the highest values in ethanol (200 µg L−1). This volatile compound can accumulate in very high concentrations in mandarins due to the fermentation process caused by a lack of oxygen [12]. In addition, the rootstock “Forner-Alcaide 517” stood out together with “Carrizo citrange” for its high content in limonene (12,785 and 12,278 µg L−1, respectively), myrcene (485 and 521 µg L−1, respectively), linalool (323 and 416 µg L−1, respectively), octanal (171 and 149 µg L−1, respectively), and 1-octanol (63.3 and 59.0 µg L−1, respectively). Several authors include limonene, linalool, terpinene-4-ol (wood), and myrcene as key aroma volatile compounds in mandarin juice [8,12,19,26,27]. Furthermore, α-pinene is considered a positive contributor to citrus fruits aroma [22,28]. In this case, “Carrizo citrange” and “Forner-Alcaide 517” showed the highest values of this volatile compound (66.1 and 58.4 µg L−1, respectively), and “Macrophylla”, “Volkameriana”, and “C-35” the lowest (13.6, 14.4, and 15.0 µg L−1, respectively). Furthermore, “Carrizo citrange” had the highest values of decanal (308 µg L−1), valencene (215 µg L−1), and terpinen-4-ol (132 µg L−1). This last compound, in certain cases, terpinen-4-ol can be considered an unpleasant aroma in mandarin fruits [21,29]. On the other hand, Chen et al. [11] found only 26 volatile compounds present in juice mandarins, with limonene being the main compound (11,617.3 µg L−1), followed by γ-terpinene (961.6 µg L−1), β-myrcene (721.9 µg L−1), α-pinene (257.7 µg L−1), and β-pinene (122.0 µg L−1). The values of these volatile compounds were higher than those found in this study. In contrast, 114 volatile compounds were found by Bai et al. [22], who identified D-limonene, β-myrcene, and α-pinene as the main compounds in citrus peel oil, and 167 aroma volatiles were identified by Yu et al. [8] in mandarin juice, including ethanol, acetone, 2-methyl-2-propanol, α-pinene, myrcene, α-terpinene, p-cymene, limonene, terpinolene, and linalool, which are present in all citrus genotypes.
These results demonstrate that rootstocks significantly affect the volatile composition of citrus. Similar results were found by Aguilar-Hernández et al. [16] in lemon fruits. In the same way, Castle [14] showed that rootstocks have effects on the quality factors of citrus fruits. The rootstocks under study were also studied by Legua et al. [17], showing their influence on the composition of bioactive constituents in mandarins. Furthermore, Saini et al. [30] found that “Kinnow” mandarin juice grafted on “Pectinifera” had the highest levels of limonene and therefore the highest values of total volatile compounds, while the same mandarin grafted on “Shekwasha” had the highest levels of β-pinene, dodecylaldehyde, octanal, α-terpineol, terpinen-4-ol, peraldehyde, nonanal, isoleucine, linalool, and hexanal. Furthermore, Raddatz-Mota et al. [31] discovered that rootstocks not only affect the volatile profile but also have an effect on the presence or absence of certain volatile compounds in the fruit. This was the case for “Persian” lime, in which β-myrcene was only found in two of the five rootstocks studied, while the compounds β-thujene and dodecane were only found in the rootstocks “Volkamer” lemon and “C-35”.
Grouping the compounds by their chemical families (Figure 1), in general, the terpenes stand out over the rest of the chemical families, being the majority in the “Carrizo citrange” and “Forner-Alcaide 517” rootstocks (Table 4). In these same rootstocks, aldehydes and alcohols were also the majority. The esters presented a higher concentration in the samples of the rootstocks “Forner-Alcaide 5” and “Forner-Alcaide 517”, while “Forner-Alcaide 418”, “Macrophylla”, and “Volkameriana” had the lowest concentrations. During the ripening of mandarins, there is an increase in the concentration of esters, which are responsible for the fruity and sweet aroma, which can lead to unpleasant aromas or the perception that the fruit is over-ripe [24]. These results agree with those obtained by Morales-Alfaro et al. [9], Benjamin et al. [15], and Cano-Lamadrid et al. [19].
Figure 1. Concentration (µg L−1) of the main chemical families identified in mandarins (Citrus clementina Hort. ex Tan.). Rootstock: “Carrizo citrange” (1), “Swingle citrumelo CPB 4475” (2), “Macrophylla” (3), “Volkameriana” (4), “Forner-Alcaide 5” (5), “Forner-Alcaide V17” (6), “C-35” (7), “Forner-Alcaide 418” (8), and “Forner-Alcaide 517” (9).
Table 4. Statistical differences found between the different chemical families. Ϯ *** significant at p < 0.001. Values followed by the same letter, within the same chemical family were not significantly different (p < 0.05), according to Tukey’s least significant difference test. Rootstock: “Carrizo citrange” (1), “Swingle citrumelo CPB 4475” (2), “Macrophylla” (3), “Volkameriana” (4), “Forner-Alcaide 5” (5), “Forner-Alcaide V17” (6), “C-35” (7), “Forner-Alcaide 418” (8), and “Forner-Alcaide 517” (9).
To gain a better understanding of the relationships established between the volatile compounds found (72), a principal component analysis (PCA) was performed on the experimental results (Figure 2). The PCA explained 68.61% of the variables, with the F1 axis being the one that explained most of the data (55.33%). The PCA showed that the rootstocks “Carrizo citrange”, “Forner-Alcaide 5”, “Forner-Alcaide 418”, and “Forner-Alcaide 517” were characterized by the most volatile compounds detected, with “Carrizo citrange” being the one that presented a different volatile profile from the other 3 rootstocks. These results agree with those obtained in the analysis of volatile compounds, in which it was these four rootstocks that presented a higher total concentration of volatile compounds. The rootstocks “Forner-Alcaide 517” and “Forner-Alcaide 5” have a common parent, so it was expected that they would present similar results [16,32].
Figure 2. A principal component analysis (PCA) plot showing the relationships among volatile compounds and the factor rootstock.

4. Conclusions

The results obtained show that the rootstock used in the cultivation of mandarins affects the volatile content of its juice. In this case, the rootstocks that showed the highest volatile concentration were “Carrizo citrange”, “Forner-Alcaide 5”, “Forner-Alcaide 418” and “Forner-Alcaide 517”, while “Macrophylla”, “Volkameriana”, and “C-35” were the least. However, more research is needed to assess the effects of the environment and other factors on rootstocks and their effect on citrus juice properties.

Author Contributions

Conceptualization, M.Á.F.-G. and P.L.; Data curation and formal analysis, P.S.-B., F.H., A.P.-C. and M.C.-L.; Funding adquisition, M.Á.F.-G.; Methodology, P.S.-B., F.H., A.P.-C., M.C.-L. and P.L.; Writing—original draft, P.S.-B., A.P.-C. and M.C.-L.; Writing—review and editing, M.Á.F.-G., P.S.-B., F.H., A.P.-C., M.C.-L. and P.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministerio de Ciencia e Innovación with the project: “Obtención, selección y evaluación de cítricos para conseguir plantaciones más sostenibles frente a las nuevas amenazas debidas al cambio global” (PID2021-124145OR-C21). Paola Sánchez-Bravo was funded by the grant for the recall of the Spanish university system for the training of young doctors (Margarita Salas, 04912/2021), funded by the European Union-Next Generation EU, Ministry of Universities of Spain.

Data Availability Statement

Data is contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

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