Changes in the Profiles of Yield , Yield Component , Oil Content , and Citral Content in Litsea cubeba ( Lour . ) Persoon Following Foliar Fertilization with Zinc and Boron

Mountain pepper (Litsea cubeba (Lour.) Persoon) is an important oil plant used as an ingredient in edible oil, cooking condiments, cosmetics, pesticides, and potential biofuels. Zinc and boron are essential micronutrients for plant growth. However, the effects of zinc and boron on the yield, yield component, oil content, and citral content in L. cubeba have not been determined. This study was conducted to evaluate the efficacy of the foliar application of zinc, boron, and multiple micronutrients (zinc + boron) on the yield, yield component, oil content, and citral content of three varieties (Fuyang 1 (FY1), Jianou 2 (JO2), and Jianou 3 (JO3)) of L. cubeba. Zinc sulfate (0.25%), boric acid (0.25%), and zinc sulfate (0.25%) + boric acid (0.25%) were sprayed on selected trees at five different times at full bloom and 28 days before harvest, once every seven days. The results indicated that Zn had a negative effect on the yield, yield component, oil content, and citral content of the FY1, JO2, and JO3 varieties compared to the untreated trees. B had positive effects on the yield, yield component, oil content, and citral content of the JO2 and JO3 varieties but not on those of the FY1 variety when compared to the untreated trees. The highest levels of yield, yield component, oil content, and citral content for all three varieties were obtained with the combined application of zinc sulfate + boric acid. Hence, the foliar application of multiple micronutrients (zinc + boron) is an effective method to improve the yield, oil content, and citral content in L. cubeba. In addition, the 100-fruit weight (HFW) was positively correlated with the yield, oil content, and citral content and could be used as a tool to select new cultivars with high yield, high oil content, and high citral content under zinc sulfate, boric acid, and zinc sulfate + boric acid applications in L. cubeba.


Introduction
Mountain pepper (Litsea cubeba (Lour.)Persoon) is an important natural aromatic plant of the Lauraceae family, which is native to southern China and is widely distributed in Southeast Asia, Japan, and Taiwan [1].All parts of this plant are rich in aromatic essential oil, and the highest essential oil content is in the fruit [2].The essential oil has been widely used as a raw material for cosmetics, pesticides, food additives, and biodiesel fuel.Meanwhile, the essential oil has various biological properties including antioxidative [3], acaricidal [4], anti-inflammatory [5], anticancer [6,7], immunosuppressive [8], fungicidal [9,10], antibacterial [10], and insecticidal [3,11,12] activities that enable its use as medicines, botanical insecticides, preservatives, and citral (neral and geranial).In addition, fruits of L. cubeba are used as a cooking condiment, which is frequently used in the

Plant Material and Treatment
Three four-year old varieties of L. cubeba, including Jianou 2 (JO2), Jianou 3 (JO3), and Fuyang 1 (FY1), were used in this experiment (Table 2).The trees were planted at 3 m × 3 m distances apart and received the same horticultural management, except in regard to fertilization with foliar treatments.Foliar fertilizer treatments were C (control, distilled water), A1 (applied ZnSO 4 0.25%), A2 (applied H 3 BO 3 0.25%), and A3 (applied ZnSO 4 0.25% + H 3 BO 3 0.25%).The trees were arranged randomly in blocks according to a factorial design Three replicates were used and each replicate contained 12 trees.The treatments were applied at five different times: at full bloom and 28 days before harvest, once every seven days.The trees were sprayed using a spraying motor (3 L capacity) until the runoff stage.The experiment was performed from March 20 to July 30 in 2017.

Preparation of Fruit Sampling
A random sample of fruits from each experimental tree was handpicked and immediately transported to the laboratory.Only fresh and healthy fruits at a technologically mature stage (this differs from the physiological maturity stage, as seeds in this stage can be used to extract oil) were used.

Determination of the Fruit Essential Oil Percentage
The oil content was extracted from the fruit samples using hydrodistillation, as described by Gao et al. [34].All samples were stored at 4 • C until analysis.

Volatile Compounds Using GC-MS Analysis
This process was almost identical to the method of Gao et al [33].The qualitative and quantitative analyses of the volatile compounds of the fruit essential oil were conducted on an Agilent 6890N/5975B gas chromatograph-mass spectrometer (GC-MS) (Agilent Technologies Co., Ltd, Palo Alto, CA, USA) using a HP-5MS fused silica capillary column (30 m × 0.25 mm internal diameter, 0.25 µm film thickness).The oven temperature was initially set at 50 • C for 2 min, then increased to 120 • C for 2 min at a rate of 3  C; ionization mode, EI; and ionization energy, 70 eV.The compound identification was confirmed by comparing the retention indices (RIs) of the samples with those reported in the literature.The RIs were calculated using a series of n-alkanes (C7-C30) under identical operating conditions.The relative amounts of the individual components were calculated using peak area normalization.In total, 47 compounds were identified from all samples.

Statistical Analysis
The data were analyzed using IBM SPSS Statistics 19 software (SPSS Inc., Chicago, IL USA).Two-way ANOVA was used to evaluate the effects of variety, fertilization, and their interaction.A Duncan multiple-comparison test was used to detect the differences between the means.A p value < 0.05 was considered significant.Pearson correlation analysis was used to analyze correlation patterns between the analyzed traits.SigmaPlot 12.0 and R3.5 (Systat Software Inc., San Jose, CA, USA) were used to draw the figures.

The Change in Yield and Yield Components during the Different Foliar Applications of Micronutrient Solutions
Analysis of variance of yield, yield components, fruit oil content, and citral contents is shown in Table 3. Different micronutrient fertilization treatments had different effects on the yield of L. cubeba (Figure 1).Zinc had a negative effect on the yield of L. cubeba varieties.The yields of the FY1, JO2, and JO3 varieties all decreased by 11.30%, 10.00%, and 17.78%, respectively, when compared to the untreated fruit.Under boric acid application, the yields in the JO2 and JO3 varieties increased by 6.67% and 22.22%, respectively, when compared to the untreated fruits.However, the yield of FY1 variety declined 3.22%.Zinc sulfate + boric acid foliar application had a positive effect on the yields of the FY1, JO2, and JO3 varieties, which were significantly increased by 45.16%, 33.33, and 33.33%, respectively, when compared to the untreated fruits.
The change in yield components during the different foliar applications of micronutrient solutions is shown in Figure 2. The same variation trend occurred between the 100-fruit weight (HFW) and yields under different micronutrient applications.The HFW of the FY1, JO2, and JO3 varieties all significantly increased (by 20.41%, 19.80%, and 41.27%, respectively) using the zinc sulfate + boric acid foliar application compared to the untreated fruits.Zn had a negative effect on the HFW of the FY1, JO2, and JO3 varieties, while B had a positive effect on the HFW of the JO2 and JO3 varieties.However, the response of the fruit longitudinal diameter (FLD), fruit transverse diameter (FTD), and fruit shape index (FSI) to the different micronutrient applications was different from those of the HFW and yield.For instance, the FLDs were all decreased with the three kinds of applications (Zn, B, Zn + B) in the    The change in yield components during the different foliar applications of micronutrient solutions is shown in Figure 2. The same variation trend occurred between the 100-fruit weight (HFW) and yields under different micronutrient applications.The HFW of the FY1, JO2, and JO3 varieties all significantly increased (by 20.41%, 19.80%, and 41.27%, respectively) using the zinc sulfate + boric acid foliar application compared to the untreated fruits.Zn had a negative effect on the HFW of the FY1, JO2, and JO3 varieties, while B had a positive effect on the HFW of the JO2 and JO3 varieties.However, the response of the fruit longitudinal diameter (FLD), fruit transverse diameter (FTD), and fruit shape index (FSI) to the different micronutrient applications was different from those of the HFW and yield.For instance, the FLDs were all decreased with the three kinds of applications (Zn, B, Zn + B) in the FY1 variety.Zn had a larger positive effect on the FLD and FTD of the JO2 variety than the other two kinds of applications (B, Zn + B).The change in yield components during the different foliar applications of micronutrient solutions is shown in Figure 2. The same variation trend occurred between the 100-fruit weight (HFW) and yields under different micronutrient applications.The HFW of the FY1, JO2, and JO3 varieties all significantly increased (by 20.41%, 19.80%, and 41.27%, respectively) using the zinc sulfate + boric acid foliar application compared to the untreated fruits.Zn had a negative effect on the HFW of the FY1, JO2, and JO3 varieties, while B had a positive effect on the HFW of the JO2 and JO3 varieties.However, the response of the fruit longitudinal diameter (FLD), fruit transverse diameter (FTD), and fruit shape index (FSI) to the different micronutrient applications was different from those of the HFW and yield.For instance, the FLDs were all decreased with the three kinds of applications (Zn, B, Zn + B) in the FY1 variety.Zn had a larger positive effect on the FLD and FTD of the JO2 variety than the other two kinds of applications (B, Zn + B).

The Change in Fruit Oil Content during the Different Foliar Applications of Micronutrient Solutions
The measurement of the fruit oil content during the different foliar applications of the micronutrient solutions is shown in Figure 3.The variation trend of the fruit oil content with different micronutrients application was consistent with that of the yields.Boric acid also had a positive impact on the fruit oil content in the JO2 and JO3 varieties, which increased 25.63% and 67.03%, respectively, when compared to the untreated fruit, except for the FY1 variety.However, the zinc sulfate treatment tended to reduce the oil content in the fruits of all three varieties when compared to the untreated fruits, and the oil content of the FY1, JO2, and JO3 varieties decreased by 25.12%, 8.00%, and 1.62%, respectively.The fruit oil content increased significantly with the Zn + B application in the FY1, JO2, and JO3 varieties and was 16.28%, 26.67%, and 103.24% higher than the untreated fruits, respectively.

The Change in Citral Contents in the Oil during the Different Foliar Applications of Micronutrient Solutions
The most dominant chemical constituent of the fruit oil in L. cubeba is citral, which constitutes approximately 80% of the chemical content of the essential oils [34].α-citral (geranial) and β-citral (neral) are cis-trans isomers of citral [37].The foliar applications of the micronutrient solutions had different effects on the chemical constituents of the different L. cubeba varieties (Figure 4).In the FY1 variety, both the Zn and B foliar sprays had negative effects on the content of neral, geranial, and

The Change in Fruit Oil Content during the Different Foliar Applications of Micronutrient Solutions
The measurement of the fruit oil content during the different foliar applications of the micronutrient solutions is shown in Figure 3.The variation trend of the fruit oil content with different micronutrients application was consistent with that of the yields.Boric acid also had a positive impact on the fruit oil content in the JO2 and JO3 varieties, which increased 25.63% and 67.03%, respectively, when compared to the untreated fruit, except for the FY1 variety.However, the zinc sulfate treatment tended to reduce the oil content in the fruits of all three varieties when compared to the untreated fruits, and the oil content of the FY1, JO2, and JO3 varieties decreased by 25.12%, 8.00%, and 1.62%, respectively.The fruit oil content increased significantly with the Zn + B application in the FY1, JO2, and JO3 varieties and was 16.28%, 26.67%, and 103.24% higher than the untreated fruits, respectively.

The Change in Fruit Oil Content during the Different Foliar Applications of Micronutrient Solutions
The measurement of the fruit oil content during the different foliar applications of the micronutrient solutions is shown in Figure 3.The variation trend of the fruit oil content with different micronutrients application was consistent with that of the yields.Boric acid also had a positive impact on the fruit oil content in the JO2 and JO3 varieties, which increased 25.63% and 67.03%, respectively, when compared to the untreated fruit, except for the FY1 variety.However, the zinc sulfate treatment tended to reduce the oil content in the fruits of all three varieties when compared to the untreated fruits, and the oil content of the FY1, JO2, and JO3 varieties decreased by 25.12%, 8.00%, and 1.62%, respectively.The fruit oil content increased significantly with the Zn + B application in the FY1, JO2, and JO3 varieties and was 16.28%, 26.67%, and 103.24% higher than the untreated fruits, respectively.

The Change in Citral Contents in the Oil during the Different Foliar Applications of Micronutrient Solutions
The most dominant chemical constituent of the fruit oil in L. cubeba is citral, which constitutes approximately 80% of the chemical content of the essential oils [34].α-citral (geranial) and β-citral (neral) are cis-trans isomers of citral [37].The foliar applications of the micronutrient solutions had different effects on the chemical constituents of the different L. cubeba varieties (Figure 4).In the FY1 variety, both the Zn and B foliar sprays had negative effects on the content of neral, geranial, and

The Change in Citral Contents in the Oil during the Different Foliar Applications of Micronutrient Solutions
The most dominant chemical constituent of the fruit oil in L. cubeba is citral, which constitutes approximately 80% of the chemical content of the essential oils [34].α-citral (geranial) and β-citral (neral) are cis-trans isomers of citral [37].The foliar applications of the micronutrient solutions had different effects on the chemical constituents of the different L. cubeba varieties (Figure 4).In the FY1 variety, both the Zn and B foliar sprays had negative effects on the content of neral, geranial, and citral.The zinc treatment decreased these three chemical constitutions when compared to the untreated fruit by 3.84%, 2.64%, and 3.17%, respectively, and the boric acid treatment decreased these three chemical constitutions from 38.31% to 36.62%, 48.03% to 45.91%, and 86.34% to 82.53%, respectively.However, Forests 2019, 10, 59 7 of 12 the contents of neral, geranial, and citral significantly increased with the B foliar spray in the JO2 and JO3 varieties, respectively.For instance, the citral contents following the boric acid treatment increased significantly from 79.33% to 82.01% and 72.31% to 85.91% in the JO2 and JO3 varieties.However, the foliar application of the compound nutrients (Zn + B) increased three chemical constituent contents in all three varieties.For example, the citral content increased by 0.27%, 7.37%, and 23.58%, respectively, when compared to that of the untreated fruits.
citral.The zinc treatment decreased these three chemical constitutions when compared to the untreated fruit by 3.84%, 2.64%, and 3.17%, respectively, and the boric acid treatment decreased these three chemical constitutions from 38.31% to 36.62%, 48.03% to 45.91%, and 86.34% to 82.53%, respectively.However, the contents of neral, geranial, and citral significantly increased with the B foliar spray in the JO2 and JO3 varieties, respectively.For instance, the citral contents following the boric acid treatment increased significantly from 79.33% to 82.01% and 72.31% to 85.91% in the JO2 and JO3 varieties.However, the foliar application of the compound nutrients (Zn + B) increased three chemical constituent contents in all three varieties.For example, the citral content increased by 0.27%, 7.37%, and 23.58%, respectively, when compared to that of the untreated fruits.

Correlations
Under the zinc sulfate foliar application, the yield was positively correlated with the HFW, oil content, neral, geranial, and citral (Figure 5a).In addition, there were positive correlations between the HFW, oil content, neral, geranial, and citral.Under boric acid foliar application, the yield was positively correlated with the FLD, FSI, and HFW (Figure 5b).In addition, the oil content was positively correlated with the FTD and HFW.The four indicators of HFW, neral, geranial, and citral were positively correlated with each other.Under the zinc sulfate + boric acid foliar applications, the yield was positively correlated with the HFW and oil content (Figure 5c).In addition, the five indicators of HFW, oil content, neral, geranial, and citral had positive correlations with each other.

Correlations
Under the zinc sulfate foliar application, the yield was positively correlated with the HFW, oil content, neral, geranial, and citral (Figure 5a).In addition, there were positive correlations between the HFW, oil content, neral, geranial, and citral.Under boric acid foliar application, the yield was positively correlated with the FLD, FSI, and HFW (Figure 5b).In addition, the oil content was positively correlated with the FTD and HFW.The four indicators of HFW, neral, geranial, and citral were positively correlated with each other.Under the zinc sulfate + boric acid foliar applications, the yield was positively correlated with the HFW and oil content (Figure 5c).In addition, the five indicators of HFW, oil content, neral, geranial, and citral had positive correlations with each other.

The Effect of Zn and B Foliar Applications on the Yield and Yield Components in L. cubeba
The yields of the FY1, JO2, and JO3 varieties all decreased under zinc application, which suggested that the zinc fertilizer application had a negative effect on the yield of L. cubeba with 6.1 mg/kg of available zinc content in the soil at a depth of 0-30 cm in our experiment.This finding was consistent with those of Wang et al. [28], Zhang et al. [38], and Wang et al. [32].The capacity of the soil zinc supply was divided into five levels according to the available Zn content in China, and the available Zn content of 1.5 mg/kg was used for the threshold of the soil Zn deficiency [39].This indicates that the soil in this experiment cannot be classified as deficient in Zn, and therefore, the available Zn in the soil is not low enough to show a yield increase by applying additional Zn [28].In addition, the fact that the yield did not increase with the Zn application could be due to the application of the treatments during the reproductive vegetative stage and not during the seedling stage.Some previous studies demonstrated that plant growth and yield did not increase with late Zn application [28,40,41].The root Forests 2019, 10, 59 9 of 12 and shoot growths and the yield of the plants from high-Zn seeds were markedly greater than those from low-Zn seeds [42,43].For the particular case of L. cubeba, the effect of foliar fertilization of zinc in the growing period is unknown, which should be determined in future research.
In China, soil available boron content of 0.5 mg/kg is the critical threshold of soil boron deficiency, and soil available boron content between 0.5 and 1.0 mg/kg is a medium level [44].In this case, the yield would increase with boron application.In our research, the available boron content of the soil at a depth of 0-30 cm was 0.8 mg/kg and 0.4 mg/kg at a depth of 30-60 cm.L. cubeba is a shallow-rooted plant, and the depth of the root is above 25 cm.Hence, the boron application was available to the trees in our study.However, the yield of the FY1 variety was not affected by boron application, which is likely because the effect of the boron application also depends on a different genotype.Therefore, breeders should use available genetic diversity to enhance yield [45].In addition, foliar sprays of B may be used to address a temporary deficiency, but to achieve full growth and development, B must be applied throughout the plant cycle [24].
In our experiment, the highest yields of all three varieties were obtained by the zinc sulfate + boric acid foliar application.This is likely because the effect of foliar application of compound nutrients was better than that of single micronutrients to increase the oil content of L. cubeba.
Yield components are very important because they are related to the fruit yield [14].In L. cubeba, one of the most important yield components is the HFW, which is significantly positively correlated to the fruit yield, but there was no relation among the FLD, FTD, FSI, and yield [33].Our results showed that the effect of micronutrient application on HFW was more than the effect on FLD, FTD, and FSI.

The Effect of Zn and B Foliar Applications on the Oil Content and Citral Contents in L. cubeba
In our study, the oil contents of all three varieties were decreased under zinc sulfate treatment.However, the reports of Saadati et al. and Ramezani et al. reported higher oil contents in olive cultivar fruits following foliar zinc application [29,46].This might indicate that different plants have different sensitivities to micronutrients [40].Maize, sorghum, and soybean are very sensitive to zinc, but wheat, barley, and carrot are not.At the same time, several factors also limit the efficiency of the micronutrients on oil content, including soil condition, environment, time of application, and the background nutrient status of the soil and plants [32].
These findings were consistent with those of Desouky et al. and Saadati et al. [27,29].Desouky et al. reported that the foliar application of different micronutrients on olive cultivars could manipulate the compositions of fatty acids differently.In addition, variations in the oil chemical constitution during the foliar application of the nutrient solutions observed in the L. cubeba varieties could be related to both genetic factors and environmental conditions [47].
4.3.The Tool to Select New Cultivars with High Yield, High Oil Content, and High Citral Content under Zn and B Foliar Applications L. cubeba is a species that has not been studied extensively, and its responses to different micronutrient treatments (e.g., zinc sulfate and boric acid) are unknown.This is the first report on the effect of zinc sulfate and boric acid supply on the yields, yield components, oil contents, and citral contents, and their relationship with each other.Obviously, the HFW can be used as a tool to select new cultivars with high yield, high oil content, and high citral content under zinc sulfate, boric acid, and zinc sulfate + boric acid supplies.However, more research is needed to explore these tools for L. cubeba breeding and for better L. cubeba management, especially under other nutrient conditions.

Conclusions
Zinc and boron are essential micronutrients for all plants and are necessary for the growth and development of higher plants.We studied whether Zn and B foliar applications could affect the yield, yield component, oil content, and citral content in L. cubeba.Zn had a negative effect on the yield, yield component, oil content, and citral content of the FY1, JO2, and JO3 varieties when compared to the

ForestsFY1
variety.Zn had a larger positive effect on the FLD and FTD of the JO2 variety than the other two kinds of applications (B, Zn + B).

Figure 1 .
Figure 1.Effects of the foliar applications of the micronutrient solutions on the yield in L. cubeba (Lour.)Persoon.The different lowercase letters indicate significant differences (p < 0.05) from the different foliar applications of the nutrient solutions.Identical letters indicate the absence of significant differences.The abbreviations of "Zn", "B", "Zn + B", and "CK" represent ZnSO4, H3BO4, and ZnSO4 + H3BO4, and the control.Error bars are standard errors.The same is as below.

Figure 1 .
Figure 1.Effects of the foliar applications of the micronutrient solutions on the yield in L. cubeba (Lour.)Persoon.The different lowercase letters indicate significant differences (p < 0.05) from the different foliar applications of the nutrient solutions.Identical letters indicate the absence of significant differences.The abbreviations of "Zn", "B", "Zn + B", and "CK" represent ZnSO 4 , H 3 BO 4 , and ZnSO 4 + H 3 BO 4 , and the control.Error bars are standard errors.The same is as below.

Figure 1 .
Figure 1.Effects of the foliar applications of the micronutrient solutions on the yield in L. cubeba (Lour.)Persoon.The different lowercase letters indicate significant differences (p < 0.05) from the different foliar applications of the nutrient solutions.Identical letters indicate the absence of significant differences.The abbreviations of "Zn", "B", "Zn + B", and "CK" represent ZnSO4, H3BO4, and ZnSO4 + H3BO4, and the control.Error bars are standard errors.The same is as below.

Figure 3 .
Figure 3. Effects of the foliar applications of the micronutrient solutions on the oil content in L. cubeba.

Figure 2 .
Figure 2. Effects of the foliar applications of the micronutrient solutions on the yield components in L. cubeba.(a) HFW: 100-fruit weight; (b) FLD: fruit longitudinal diameter; (c) FTD: fruit transverse diameter; (d) FSI: fruit shape index.

Figure 2 .
Figure 2. Effects of the foliar applications of the micronutrient solutions on the yield components in L. cubeba.(a) HFW: 100-fruit weight; (b) FLD: fruit longitudinal diameter; (c) FTD: fruit transverse diameter; (d) FSI: fruit shape index.

Figure 3 .
Figure 3. Effects of the foliar applications of the micronutrient solutions on the oil content in L. cubeba.

Figure 3 .
Figure 3. Effects of the foliar applications of the micronutrient solutions on the oil content in L. cubeba.

Figure 4 .
Figure 4. Effects of the foliar applications of micronutrient solutions on the neral, geranial, and citral content in L. cubeba.The letters in black indicate significant differences of neral from the different foliar applications of the nutrient solutions.The letters in red indicate significant differences of geranial content from the different foliar applications of the nutrient solutions, and the letters in blue mean indicate differences of citral content from the different foliar applications of the nutrient solutions.

Figure 4 .
Figure 4. Effects of the foliar applications of micronutrient solutions on the neral, geranial, and citral content in L. cubeba.The letters in black indicate significant differences of neral from the different foliar applications of the nutrient solutions.The letters in red indicate significant differences of geranial content from the different foliar applications of the nutrient solutions, and the letters in blue mean indicate differences of citral content from the different foliar applications of the nutrient solutions.

Figure 5 .
Figure 5. Analysis of the yields, yield components, oil contents, and citral contents under zinc sulfate (a), boric acid (b), and zinc sulfate + boric acid (c) foliar applications in L. cubeba.The cells of the lower left triangle and the circles of the upper right triangle mean the same.The color in red indicates a positive correlation and blue indicates a negative correlation.The darker the color, the larger the correlation value.The correlation factors are labeled on the middle diagonal.The abbreviations "O", "N", "G," and "C" represent the oil content, neral, geranial, and citral, respectively.The symbol "*" indicates a significant correlation at the 0.05 level, and "**" indicates an extremely significant correlation at the 0.01 level.

Table 1 .
Some chemical characteristics of the experimental soil of the L. cubeba (Lour.)Persoon forest.
• C/min, and finally increased to 250 • C for 2 min at 15 • C/min.The carrier gas was N 2 at a flow rate of 1 mL/min; the injected volume was 1.0 µL (1:10 in Et 2 O), and the injector temperature was 220 • C. The other GC-MS conditions are as follows: interface temperature, 250 • C; ion source temperature, 230 • C; quadrupole temperature, 150

Table 3 .
Analysis of variance of yield, yield components, fruit oil content, and citral contents.