Effects of organic-inorganic complex fertilizer on the growth and physiological characteristics of ‘Qi-Nan’ agarwood from Aquilaria sinensis (Lour.)

Abstract

This paper is to study the effects of different levels of fertilization on the growth, physiological characteristic of ‘Qi-Nan’ agarwood seedlings. The experiment was conducted under potted conditions using a single factor experiment. Organic-inorganic complex fertilizer was used as the basal fertilizers, and 7 fertilizer gradients ranging from 0 g/plant to 23 g/plant were set up. The result showed that: (1) within a certain range, with the increase of organic-inorganic complex fertilizer application, the growth and physiological activity of ‘Qi-Nan’ had an incremental effect, but this effect would be inhibited when the fertilizer application was too high. (2) After fertilization, the contents of free proline (FP) and malondialdehyde (MDA) decreased, while the contents of osmotic regulatory substances and chlorophyll increased. (3) The correlation analysis showed that seedling height and ground diameter of ‘Qi-Nan’ seedlings were significantly positively correlated with soluble sugar content, peroxidase activity and chlorophyll content. (4) Membership function analysis showed that organic-inorganic complex fertilizer had a significant effect on the growth of ‘Qi-Nan’ seedlings, and the optimal fertilizer level of single application of organic-inorganic complex fertilizer was 7-11g/plant. Therefore, the optimal application level of organic-inorganic complex fertilizer for the seedling cultivation of ‘Qi-Nan’ was 7g/ plant.

1. Introduction

Agarwood (Chen-Xiang in Chinese), the resinous heartwood from the Aquilaria species (Thymelaeaceae) [1], has widely used in medicine, aromatherapy, perfume industry, religious culture, and other fields. It is a valuable traditional medicine and natural spice [2–4]. ‘Qi-Nan’, also known as Kanankoh, Kyara [5,6] or Chi-Nan [7]. The wild ‘Qi-Nan’ is usually considered to be the highest quality wild agarwood wood, and its price is thousands of times higher than ordinary agarwood [8]. However, due to the destruction of the natural environment and excessive deforestation caused by economic interests, wild Aquilaria species are endangered [9]. This genus is now listed in Appendix II of the Convention on Internal Trade in Endangered Species of Wild Fauna and Flora [10]. Additionally, the low yield of wild Aquilaria species cannot meet the market demand. Therefore, planting agarwood trees have become an important way to obtain agarwood. In recent years, grafted ‘Qi-Nan’ agarwood tree has emerged. It is a high-quality agarwood obtained by grafting and breeding from natural wild agarwood trees [11,12]. Aquilaria sinensis “Reke2” is an example of high-quality cultivated ‘Qi-Nan’ propagated by grafting with the ordinary germplasm of Aquilaria sinensis [13]. It has been cultivated in places such as Hainan, Guangdong, Guangxi, and Yunnan, with excellent results. Compared to trees grown from ordinary agarwood seedlings, grafted ‘Qi-Nan’ agarwood can more easily produce higher-quality agarwood and has the advantages of early incense-bearing and high yield [11,14]. In order to satisfy the demand of the agarwood market, ‘Qi-Nan’ agarwood is being vigorously promoted and planted in China. According to preliminary statistics, 5,766 acres of ‘Qi-Nan’ agarwood seedlings have been planted nationwide [15–17]. Fertilization is a key measure for seedling planting [18] and is essential for the healthy growth of trees, which is the basis for forming more agarwood.

China’s traditional crops and forestry on the planting of chemical fertilizer used in a single species, organic fertilizer accounted for a low proportion of fertilizer nutrient distribution ratio unreasonable is an important reason for the low utilization of fertilizer, and over-fertilization will cause a series of environmental pollution problems [19]. Some studies have pointed out that under the same nutrient content conditions, the nutritional effect of organic fertilizers applied in combination with chemical fertilizers exceeds that of single application of chemical fertilizers or single application of organic fertilizers, and at the same time, it plays a role in promoting the mineralization of organic fertilizers, activating the phosphorus in the soil, reducing the fixation of inorganic phosphorus, and extending the fertilizer supplying performance of chemical nitrogen fertilizers [20]. The application of organic-inorganic complex fertilizer is mostly seen in crops [14, 21–23], and its application has reached a certain scale, and the related research has also reached a certain degree. However, the application of organic-inorganic fertilizers on forest trees is relatively small, only focusing on the application of common tree species, such as Cinnamomum camphora [24], Larix gmelinii [25], Eucalyptus [26], etc. The application of organic-inorganic complex fertilizer on agarwood is rarely researched, especially on such precious tree species as ‘Qi-Nan’ agarwood.

At the fertilization level of agarwood plants, some scholars have studied the fertilization methods of agarwood. For example, the combination application of 10 g biological bacterial fertilizer and 20 g complex fertilizer has the best effect on promoting the growth of Aquilaria sinensis seedlings and enhancing soil fertility [27]. The use of a combination of inorganic fertilizer, organic fertilizer and biofertilizer treatments can better promote the growth of agarwood seedlings [28]. In addition, the fertilization method of Aquilaria malaccensis was also preliminarily explored. The photosynthetic capacity, growth amount and biomass of Aquilaria malaccensis were maximized when N was applied at 3 000 mg/plant. And when N was applied at 3.637g/ plant, P was applied at 2.5g/ plant and K was applied at 1.25g/ plant, the maximum biomass of Aquilaria malaccensis could be obtained at 27.688 g [29]. At present, however, there is a lack of studies on the fertilization and growth of ‘Qi-Nan’ seedlings. For example, the effects of different fertilization level on the growth and physiology of grafted ‘Qi-Nan’ agarwood seedlings have not been reported. The background that the quality and yield of ‘Qi-Nan’ agarwood are far from meeting the market demand, how to adopt a reasonable fertilization mode to achieve efficient seedling breeding is a significant problem; this problem needs to be solved urgently in the current cultivation process of ‘Qi-Nan’ agarwood seedlings.

Therefore, in this study, organic-inorganic complex fertilizer was used as base fertilizers to study the effects on the growth and physiological characteristics of ‘Qi-Nan’ agarwood seedlings. The purpose of this study was threefold: (1) to determine the changes of the growth and physiology of ‘Qi-Nan’ seedlings under different fertilizing amounts; (2) to determine which physiological and nutrient indexes were the key factors affecting the height and ground diameter of ‘Qi-Nan’ seedlings; (3) to explore the level of organic-inorganic complex fertilizer application that is most conducive to the growth and development of ‘Qi-Nan’ agarwood seedlings.

2. Materials and methods

2.1. Experimental materials

The experimental varieties were obtained from the seedling breeding base in Nalou Town, Nanning, Guangxi (108°63’E, 22°60’N), which has a humid subtropical monsoon climate, with an average annual rainfall of 1304.2 mm, an average annual temperature of 21.6 °C and an average annual relative humidity of 79% [30]. Grafted ‘Qi-Nan’ agarwood seedlings with healthy and consistent growth for 2 months were selected as experimental materials in October 2021. The average seedling height was 17.60 ±  0.33 cm (average ±  standard deviation) and the average ground diameter was 4.95 ±  0.29 mm (average ±  standard deviation). The roots of the seedlings were kept clean before transplanting and transplanted into a plastic pot (with a tray) with a diameter of 264 mm and a height of 239 mm. The local yellow loam and sandy soil were mixed 1:1 as potting soil, and each pot was filled with 0.5 kg soil. The background values of the soil were measured before the experiment. The pH was 4.07, the alkali-hydrolyzed nitrogen was 29.43 mg/kg, the available potassium was 77.69 mg/kg, the available phosphorus was 30.14 mg/kg, and the organic matter content was 17.1 g/kg. The fertilizer used in the test was potassium sulfate compound fertilizer, produced by Miaoran Agricultural Chemical Technology Co., LTD., with total nitrogen ≥  15%, total potassium ≥  15%, total phosphorus ≥  15%, and total nutrient content ≥  45%.

2.2. Experiment design

The experiment was a single factor experiment of organic-inorganic complex fertilizer. 7 levels of organic-inorganic complex fertilizer were set to fertilize the current year’s sown seedlings of ‘Qi-Nan’ agarwood. A total of 7 treatments were set up, CK (0 g/ plant), 1 (3 g/ plant), 2 (7 g/ plant), 3 (11 g/ plant), 4 (15 g/ plant), 5 (19 g/ plant), 6 (23 g/ plant). Each treatment was 12 plants with 3 replications. After a month of rejuvenation period, the grafted ‘Qi-Nan’ agarwood seedlings were fertilized. The background values of seedling height and ground diameter were measured before fertilization. All fertilization treatments were completely consistent in the management of supply, weeding, pest control and so on during the experiment, to minimize the experimental errors in the process of fertilization treatment. Fertilizer was applied twice, in November 2021 and May 2022, respectively, because of the seasonal nutrient uptake pattern of seedlings considered in the pot experiment. Evenly sprinkle the corresponding amount of organic and inorganic complex fertilizer on the surface of the potting soil, and then water when fertilizing. The experiment was conducted from November 2021 to November 2022, and the experimental period was one year.

2.3. Sample collection and determination

2.3.1. Determination of growth index.

The plant height and ground diameter of ‘Qi-Nan’ agarwood seedlings were measured with steel tape (accuracy of 0.1 cm) and vernier caliper (accuracy of 0.1mm), respectively. The measurements were taken from November 2021 to November 2022, and the heights and ground diameters of all potted seedlings were measured every other month for a total of 12 times, each at the exact location.

2.3.2. Measurement of physiological indexes and leaf nutrient content.

For each treatment, three well-grown seedlings were randomly selected, and the upper branches were used to count the 2nd to 5th mature functional leaves from the terminal bud downwards. These leaves were wiped clean, put into plastic bags, labeled, and temporarily stored in ice boxes; after collection, they were brought back to the laboratory and stored in an ultra-low-temperature refrigerator (-70 ^◦C). The contents of chlorophyll, soluble sugar, soluble protein, free proline, and malondialdehyde were determined by the acetone extraction method [31], anthrone colorimetric method [32], Coomassie brilliant blue method [33], acid ninhydrin colorimetric method [34], and thiobarbituric acid colorimetric method [32], respectively. The activities of superoxide dismutase and peroxidase were determined by acid ninhydrin colorimetry [35] and guaiacol colorimetry [32]. The measurement time was February 2022, May 2022, August 2022, and November 2022, with a total of 4 measurements.

2.3.3. Data analysis and processing methods.

The data were organized by Excel 2016; The SPSS 25.0 software (SPSS Inc., Chicago, IL, USA) was used to perform the statistical analyses by ANOVA and significance tests. p ≤  0.05 was considered to indicate statistically significant differences. Plotting was performed by origin 2021; The membership function method of fuzzy mathematics was used for the comprehensive evaluation of the growth and physiological indexes of ‘Qi-Nan’ agarwood under the effect of organic-inorganic complex fertilizer.

3. Results

3.1. Effects of organic-inorganic complex fertilizer on growth indexes of ‘Qi-Nan’ agarwood seedlings

3.1.1. Effects of organic-inorganic complex fertilizer on seedling height of ‘Qi-Nan’ agarwood seedlings.

From Fig 1 that under different fertilization treatments, the dynamic change of plant height growth of ‘Qi-Nan’ agarwood seedlings showed an upward trend, and the growth rhythm was characterized by “slow-fast-slow” and showed an S-shaped curve. After May of the following year, the seedlings entered the rapid growth period, the seedling height growth increased significantly, in which the height growth of the seedlings was extremely rapid from June to August, and they entered the peak period of seedling height growth.

[Figure omitted. See PDF.]

From Table 1, the differences in seedling height between groups of ‘Qi-Nan’ agarwood seedlings at the end of fertilization were significant (P <  0.05). The results of multivariate analysis of variance showed that different amounts of organic-inorganic complex fertilizer treatment and different growth months had significant effects on plant height growth (P <  0.01). Among them, the seedling height and total growth of ‘Qi-Nan’ agarwood seedlings were the largest in treat 2, 81.8 cm and 64.7 cm, respectively, which were 144.5% and 167.3% of CK, followed by treat 3. The seedling height and total growth of ‘Qi-Nan’ agarwood in treat 4 were the smallest, 65.1 cm and 47.3 cm, respectively, which were 115.0% and 122.5% of CK.

[Figure omitted. See PDF.]

Multiple comparisons of treatment can be seen from Table 2. As the concentration of organic-inorganic complex fertilizer increases, it is helpful for the height growth of ‘Qi-Nan’ agarwood. The seedling height growth of treatment 2 is the largest, followed by treatment 3, and then the seedling height growth gradually decreases with the increase of fertilization concentration. The concentration of organic-inorganic complex fertilizer that can promote the seedling height ranges from 7-11g/ plant.

[Figure omitted. See PDF.]

The multiple comparisons of the main effect of months in Table 3 show that the monthly growth of seedling height first increases and then decreases with the fertilization time. Among them, the differences between each month are extremely significant, and the growth of seedling height is the largest in August.

[Figure omitted. See PDF.]

In conclusion, different concentrations of organic and inorganic compound fertilizer have significant differences on seedling height, and the optimal concentration of organic and inorganic compound fertilizer topdressing for seedling height growth of ‘Qi-Nan’ agarwood is about 7-11g/ plant.

3.1.2. Effect of organic-inorganic complex fertilizer on the ground diameter of ‘Qi-Nan’ agarwood seedlings.

From Fig 2 that in the early stage of fertilization (2021.11-2022.2), the effect of each treatment on the growth of ‘Qi-Nan’ incense was not obvious, and the growth of seedling diameter was still slow in March and April. From May to June is the peak period of the growth of the ground diameter, and the growth of the ground diameter in all treatments is larger in June. After June, the growth rate decreased, and the increase in diameter of each treatment was more average in different months from June to October.

[Figure omitted. See PDF.]

From Table 4, after fertilization, the seedling height and ground diameter of ‘Qi-Nan’ agarwood seedlings had significant differences among groups (P <  0.05). The results of multivariate analysis of variance showed that different amounts of organic-inorganic complex fertilizer treatment and different growth months on plant height growth were highly significant (P <  0.01). Among them, treat 2 had the largest growth in diameter and total diameter, which were 160.38% and 214.24% of CK, followed by treat 3. Treat 6 had the smallest growth in diameter and total diameter, which were 0.83 mm and 0.33 mm less than that of the CK, and some of the seedlings in treat 6 were dead, which may be due to ‘burnt’ caused by the application of the high-concentration fertilizers.

[Figure omitted. See PDF.]

From Table 5, multiple comparisons of the main effects indicated that with the increase in the topdressing amount of organic-inorganic compound fertilizers, the ground diameter growth of ‘Qi-Nan’ agarwood seedlings initially ascended and then declined. A high concentration of organic-inorganic compound fertilizers inhibited the growth of seedlings and even severely hampered the growth of seedling diameter. Among them, the seedling diameter growth was the highest under treatment 2, suggesting that a topdressing amount of 7g/plant was more conducive to the seedling diameter growth of ‘Qi-Nan’ agarwood.

[Figure omitted. See PDF.]

From Table 6, the monthly growth of the ground diameter displayed a trend of initially rising and subsequently falling with the fertilization time. There was no significant difference between February and May, yet a highly significant difference occurred between August and November. The disparity between August and November was also highly significant. These results imply that the growth of the ground diameter accelerates in August, then slows down and essentially stops in November.

[Figure omitted. See PDF.]

In conclusion, the effects of different concentrations of organic and inorganic compound fertilizer on the soil diameter are significantly distinct, and the optimal concentration of organic and inorganic compound fertilizer for the soil diameter growth of ‘Qi-Nan’ agarwood seedlings is approximately 7g/plant.

3.2. Effects of organic-inorganic complex fertilizer on physiological and biochemical indexes of ‘Qi-Nan’ agarwood seedlings

3.2.1. Effects of organic-inorganic complex fertilizer on the content of Osmotic mediating substances in ‘Qi-Nan’ agarwood seedlings.

According to the results of the effects on soluble protein (SP) (Fig 3a), the SP content of ‘Qi-Nan’ agarwood seedlings differed between groups (P <  0.05) in February, May, August and November. The results of multivariate analysis of variance showed that the effects of different fertilizer amount of organic-inorganic complex fertilizer treatments and different growth months on SP content were highly significant (P <  0.01). Overall, the SP content of ‘Qi-Nan’ agarwood seedlings did not change significantly over time under different amounts of organic and inorganic compound fertilizer. With the increase of fertilizer application, the SP content generally increased first and then decreased. Except that the SP content of treat 2 in November was lower than that of treat 3, the SP content of treat 2 and treat 3 in other months was higher, indicating that the two concentration levels of fertilization had a better effect on the synthesis of SP content in seedlings. Within a certain range, the SP content of ‘Qi-Nan’ agarwood seedlings also increased with the increase of the amount of organic-inorganic complex fertilizer application, but the SP content would decrease beyond a certain concentration.

[Figure omitted. See PDF.]

(a) soluble protein; (b) soluble sugar. Different capital letters indicate highly significant differences between the same index, the same period, and different treatments at the 0.01 level (P < 0.01), and different lowercase letters indicate significant differences between the same index, the same period, and different treatments at the 0.05 level (P < 0.05).

According to the results of the effect on soluble sugar (SS) (Fig 3b), the SS content of ‘Qi-Nan’ agarwood seedlings differed between groups (P <  0.05) in February, May, August and November. The results of multivariate analysis of variance showed that the effects of different amounts of organic-inorganic compound fertilizer treatment and different growth months on SS content were highly significant (P <  0.01). Overall, the SS content of ‘Qi-Nan’ agarwood seedlings showed an increasing trend over time, and the overall change was small. The SS content in February increased first and then decreased with the increase of fertilizer application. In August, the SS content of treat 3 and 4 was lower than that of CK, but entered November, the SS content showed an upward trend with the increase of fertilizer application, and the SS content of each fertilizer treatment was higher than that of CK, indicating that organic and inorganic compound fertilizer had a better promotion effect on the synthesis of SS. Meanwhile, the SS content of treat 6 reached the maximum.

3.2.2. Effect of bio-organic fertilizer treatments on enzyme activity of ‘Qi-Nan’ agarwood seedlings.

From the results of the effect on peroxidase (POD) activity (Fig 4a), there were differences (P <  0.05) between groups of POD activity in ‘Qi-Nan’ seedlings in February, May, August and November. The results of multifactorial ANOVA showed that the effects of different fertilizer doses of organic and inorganic composite fertilizer treatments and different growth months on POD activity were highly significant (P <  0.01). Under different fertilization treatments of organic and inorganic compound fertilizers, the POD activity of ‘Qi-Nan’ seedlings showed an increasing and then decreasing trend with the increase of fertilization. Throughout the growth process, POD activity showed a general upward trend with time, indicating that peroxidase activity was weak in young seedlings and high in mature seedlings. POD activity was higher in all fertilization treatments than in CK. During the month of November, the POD activity of treatments 1, 2, and 3 increased compared to August, whereas treatments 4, 5, and 6 decreased compared to August, indicating that the POD activity continued to increase at low concentrations, and was higher in the early part of the year and lower in the later part of the year at high concentrations.

[Figure omitted. See PDF.]

(a) peroxidase; (b) superoxide dismutase. Different capital letters indicate highly significant differences between the same index, the same period, and different treatments at the 0.01 level (P < 0.01), and different lowercase letters indicate significant differences between the same index, the same period, and different treatments at the 0.05 level (P < 0.05).

From the results of the effect on superoxide dismutase (SOD) activity (Fig 4b), there were differences (P <  0.05) between groups of SOD activity in ‘Qi-Nan’ seedlings in February, May, August and November. The results of multifactorial ANOVA showed that the effects of different fertilizer application rates of organic-inorganic composite fertilizer treatments and different growing months on SOD activity were highly significant (P <  0.01). The SOD activity of ‘Qi-Nan’ seedlings increased with the increase of organic and inorganic composite fertilizers in each fertilization treatment. In February and May, the SOD activity of treatment 6 was high compared to that of treatments CK, 1, 2, and 3 with low concentration, while in November it was low compared to that of treatments with low concentration, indicating that superoxide dismutase activity was higher in the early stage of the treatment with high fertilizer concentration, and declined or flat in the later stage of the treatment; the SOD activities of the control CK and the treatments 1, 2, and 3 with low concentration largely showed an increase with the increase of the concentration throughout the growth process, and the SOD activeness could be maintained with a sustained growth in the low concentration.

3.2.3. Effect of fertilization on malondialdehyde in ‘Qi-Nan’ seedlings.

From the results of the effect on malondialdehyde (MDA) (Fig 5), there were differences (P <  0.05) between groups in the MDA content of ‘Qi-Nan’ seedlings in February, May, August and November. The results of multifactorial ANOVA showed that the effects of different fertilizer application rates of organic-inorganic composite fertilizer treatments and different growing months on MDA content were highly significant (P < 0.01). The MDA content of ‘Qi-Nan’ seedlings varied greatly among different fertilizer treatments and different months throughout the growth process. The changes in MDA content of ‘Qi-Nan’ seedlings treated with different fertilization rates in different months were basically the same, generally showing a decreasing and then increasing trend. The MDA content of the treatments was comparable in February and November, both of which were significantly higher than in May and August, while the lowest MDA content was found in all treatments in August.

[Figure omitted. See PDF.]

Different capital letters indicate highly significant differences between the same index, the same period, and different treatments at the 0.01 level (P < 0.01), and different lowercase letters indicate significant differences between the same index, the same period, and different treatments at the 0.05 level (P < 0.05).

3.2.4. Effects of fertilization on chlorophyll content of ‘Qi-Nan’ seedlings.

From the results of the effect on chlorophyll (Chl) content (Fig 6), the Chl content of ‘Qi-Nan’ seedlings differed between groups (P <  0.05) in February, May, August and November. The results of multifactorial ANOVA showed that the effects of different fertilizer rates of organic-inorganic composite fertilizer treatments and different growing months on Chl content were highly significant (P <  0.01). Overall, it appeared that the Chl content of ‘Qi-Nan’ seedlings increased and then decreased with the time of fertilization, reaching a maximum in August, when photosynthesis was strongest. Except for treatment 6, the Chl content of all treatments was higher than that of CK, indicating that organic and inorganic composite fertilizers can effectively promote the growth of Chl within a certain range of fertilizer application. The Chl content of treatment 6 was lower than that of CK in the periods of February, May, and August, and slightly higher than that of CK in November, indicating that the application of high concentration of organic and inorganic compound fertilizer in the early stage exerted a stressful effect on the ‘Qi-Nan’ sedum seedling, which inhibited Chl synthesis in the seedling. As the root system of the seedlings grew later in the season, the seedlings became more resilient, allowing the chlorophyll content to be gradually higher than that of CK.

[Figure omitted. See PDF.]

Different capital letters indicate highly significant differences between the same index, the same period, and different treatments at the 0.01 level (P < 0.01), and different lowercase letters indicate significant differences between the same index, the same period, and different treatments at the 0.05 level (P < 0.05).

4. Comprehensive analysis of the indicators of ‘Qi-Nan’ seedlings

Correlation analysis of different growth physiological indexes in each month is shown in Fig 7. Seedling height was highly significantly positively correlated with SS and POD, and significantly positively correlated with Chl; Ground diameter was highly significantly and positively correlated with SS, Chl and POD; POD showed highly significant positive correlation with SS, significant positive correlation with SS, significant positive correlation with Chl and significant negative correlation with MDA; MDA showed highly significant negative correlation with Chl and SOD. This indicates that when the Chl content, SS content and POD activity are higher, the seedling height and diameter of ‘Qi-Nan’ seedlings will grow better; on the contrary, when the Chl content, SS content and POD activity are lower and the MDA content is higher, the seedling height and diameter of ‘Qi-Nan’ seedlings will grow worse.

[Figure omitted. See PDF.]

(a) plant height (PlH); (b) ground diameter (GrD); (c) soluble protein (SP) (d) soluble sugar (SS); (e) Free proline (FP); (f) superoxide dismutase (SOD); (g) peroxidase (POD); (h) malondialdehyde (MDA); (i) chlorophyll (Chl). p<0.5: *; p<0.1: **; p<0.01: ***.

Based on the results of the above growth and physiological condition tests, membership function analysis was used to synthesize and analyze the results in Table 7. Based on the mean value of affiliation, it can be concluded that treatment 3 has the highest composite score, followed by treatment 2, treatment 5 and treatment 4. The composite ranking is 3 >  2 >  5 >  4 >  1 >  6>  CK.

[Figure omitted. See PDF.]

Among them, the score of treatment 5 is higher than that of treatment 4, which may be due to the fact that the malondialdehyde score of treatment 5 is much larger than that of treatment 4, while the scores of the other indicators do not differ much, which makes the mean value of the affiliation of each indicator of treatment 5 larger than that of treatment 4. It showed that treatment 5 was subjected to greater stress and produced more malondialdehyde than treatment 4; treatments 4 and 5 scored higher than treatments 1 and 6, indicating that treatment 6 was over-fertilized compared to treatments 4 and 5, and that treatment 1 was under-fertilized compared to treatments 4 and 5, and that over- or under-fertilizing was detrimental to the growth of seedlings; and treatment 1 scored higher than treatment 6, suggesting that organic and inorganic composite fertilizers at high concentrations inhibited the growth of seedlings.

5. Discussion

5.1. Effects of organic and inorganic compound fertilizer on growth indexes of agarwood seedlings of ‘Qi-Nan’

Plant height, ground diameter and so on, as the key growth indicators of forest growth, can well reflect the growth state of forest [36]. The seedling height and diameter were higher than CK in all fertilizer treatment groups in this experiment, except for treatment 6, which had a slightly smaller diameter than CK. Among them, the seedling height and ground diameter of treatment 2 reached the maximum among the groups, which were 44.52% and 60.38% higher than CK, respectively. This suggests that moderate application of organic-inorganic complex fertilizers will promote the growth of ‘Qi-Nan’ seedlings, which is similar to the findings of olive tree [37], Aquilaria sinensis [28], Aquilaria yunnanensis [38] and Corylus heterophylla [39]. However, fertilization should be appropriate, too high or too low fertilization are not conducive to the growth of seedlings, too low fertilization cannot provide seedlings with sufficient nutrients, while too high fertilization may cause seedlings of the “seedling burning” phenomenon, the seedlings have a toxic effect. In this experiment, under the treatment of 7-11g/plant of organic-inorganic complex fertilizer, the growth performance of ‘Qi-Nan’ seedlings were optimal, and the growth condition of the treatment group with more than 11g/plant became worse gradually with the increase of fertilizer application. This indicates that within a certain range of fertilizer application, a reasonable amount of fertilizer will significantly promote seedling growth, while too high or too low fertilizer application will be detrimental to seedling’s growth. This is consistent with the results of the study of Sapindus mukorossi [40] and Amorpha fruticose [41] seedlings. With the increase of fertilizer application, the growth indicators of Sapindus mukorossi and Amorpha fruticose showed a tendency of increasing and then decreasing. In addition, the growth trend of seedlings in the whole stage is “slow, fast and slow”, which may be attributed to the weak root system of seedlings in the early stage of growth, the application of organic-inorganic complex fertilizers and the cold weather in winter produced a certain degree of stress on the plant, which requires a period of slowing down the seedling period.

5.2. Effects of organic and inorganic fertilizers on the physiology of ‘Qi-Nan’ seedlings

The effects of different concentrations of organic-inorganic complex fertilizers on the physiological indexes of ‘Qi-Nan’ seedlings varied. In this experiment, the chlorophyll content, soluble protein content, and POD content of seedlings showed a trend of increasing and then decreasing with the increase in the amount of fertilizer, which is consistent with the results of different nitrogen, phosphorus, and potassium application on buckwheat leaves [42], and different concentrations of water-fertilizer coupling on greenhouse bag-grown tomatoes [43]. This suggests that a reasonable amount of fertilizer will improve the photosynthetic capacity of ‘Qi-Nan’ seedlings and promote plant carbon and nitrogen metabolism. SS and SP are both indispensable nutrients in plants, and they also play an important role in maintaining osmoregulation in plants [44]. When the concentration of organic-inorganic complex fertilizers was too high, the SS content kept increasing, indicating that the high concentration of organic-inorganic complex fertilizers promoted the SS content of ‘Qi-Nan’ seedlings better, while the content of SP appeared to be decreasing instead. The reason may be that the high concentration of fertilizer caused high protease activity in ‘Qi-Nan’ seedlings, which accelerated the rate of protein hydrolysis, while RNA transcription and translation were inhibited [45], resulting in a decrease in the SP content in ‘Qi-Nan’ seedlings. This is in line with the results emerging from the study of physiological and ecological adaptation of Suaeda heteropptera Kitagawa to salt stress alleviation [46]. FP and MDA are indicators of stress tolerance in plants [47], and with the increase of o organic-inorganic complex fertilizer application, the content of MDA and FP first decreased and then increased, indicating that the appropriate concentration of organic-inorganic complex fertilizer can reduce the environmental stress suffered by ‘Qi-Nan’ seedlings. Under adverse conditions, the metabolism of reactive oxygen species in plants is imbalanced with the scavenging of the defense system and the membrane structure is disrupted [48]. In contrast, POD and SOD, as protective enzyme systems in plants, are used to remove reactive oxygen species free radicals, relieve membrane lipid peroxidation of cells, and reduce the damage to plants caused by the hostile environment [49]. In the experiment, the difference of enzyme activity between different concentrations of organic-inorganic complex fertilizers was highly significant. From the point of view of the activity size, if the concentration was too low, the promotion of enzyme activity in ‘Qi-Nan’ seedlings was not obvious, and if the concentration was too high, the enzyme activity was inhibited by the fertilizers, so the reasonable amount of organic-inorganic complex fertilizers could help to increase the enzyme activity in ‘Qi-Nan’ seedlings and enhance the seedling’s resistance.

6. Conclusions

‘Qi-Nan’ agarwood is the highest quality and value agarwood on the market. However, no one has studied the suitable fertilization method for potted seedlings. Our study found that organic-inorganic complex fertilizer has a greater effect on the growth and development of ‘Qi-Nan’ seedlings, and that moderate amounts of organic-inorganic complex fertilizers will promote the growth of ‘Qi-Nan’ seedlings, while excessive amounts will have an inhibitory effect on their growth. Correlation analysis showed that the main factors affecting the seedlings’ heights and diameters were POD, chlorophyll, and SS content, and all three of them had higher content around November, indicating that ‘Qi-Nan’ potted seedlings had strong photosynthetic capacity and better growth in November. Overall, the optimal fertilizer amount of ‘Qi-Nan’ agarwood potted seedlings is about 7-11g/ plant. However, the results obtained from the pot trials were limited and not sufficiently representative of long-term fertilization effects. Therefore, it is recommended to follow up with additional field trials of ‘Qi-Nan’ fertilization and continue the research so that the results of the trials will be of practical significance.

Suporting information

S1 File.

https://doi.org/10.1371/journal.pone.0320766.s001

(DOCX)

Acknowledgments

The authors would like to thank Guangxi Miaoran Agricultural Chemical Technology Co., LTD for their supply of organic-inorganic complex fertilizers, thank the agarwood cultivation base in Nalou Town for providing the environment for pot experiment, and thank relevant staff for helping to cultivate the ‘Qi-Nan’ agarwood seedlings and measuring the data.

References

1. 1. Ng LT. A review on agar (gaharu) producing Aquilaria species. J Tropic Forest Prod. 1997;2:272–85.

* View Article

* Google Scholar

2. 2. Barden A, Anak NA, Mulliken T, Song M. Heart of the matter: agarwood use and trade and CITES implementation for Aquilaria malaccensis. Traffic International. 2000;39:937–50.

* View Article

* Google Scholar

3. 3. Persoon GA, van Beek HH. Growing ‘the wood of the gods’: Agarwood production in southeast Asia. Smallholder Tree Growers and Rural Development Services. 2008;2:245–62.

* View Article

* Google Scholar

4. 4. Snelder D, Rodel DL. Smallholder Tree Growing for Rural Development and Environmental Services: Lessons from Asia. Springer Science & Business Media: Berlin/Heidelberg, Germany; 2008.

5. 5. Yamagata E, Yoneda K. Pharmacognostical studies on the crude drug of agarwood (VI): On “Kanankoh”. Shoyakugaku Zasshi. 1987;41:142–6.

* View Article

* Google Scholar

6. 6. Nakanishi T, Inada A, Nishi M, Yamagata E, Yoneda K. A new and a known derivatives of 2-(2-phenylethyl) chromone from a kind of agarwood (“Kanankoh”, in Japanese) originating from Aquilaria agallocha. Journal of Natural Products. 1986;49(6):1106–8.

* View Article

* Google Scholar

7. 7. Azren PD, Lee SY, Emang D, Mohamed R. History and perspectives of induction technology for agarwood production from cultivated Aquilaria in Asia: A review. Journal of Forestry Research. 2019;30(1):1–11.

* View Article

* Google Scholar

8. 8. Naef R. The volatile and semi-volatile constituents of agarwood, the infected heartwood of Aquilaria species: A review. Flavour and Fragrance Journal. 2011;26(1):73–87.

* View Article

* Google Scholar

9. 9. Liu J, Yang J, Jiang C, Zhou J, Zhao Y, Huang L. Volatile organic compound and endogenous phytohormone characteristics during callus browning in Aquilaria sinensis. Ind Crops Prod. 2021;168:113605. Convention on Internal Trade in Endangered Species of Wild Fauna and Flora.

* View Article

* Google Scholar

10. 10. Zhang P, Li X, Cui Z, Xu D. Morphological, physiological, biochemical and molecular analyses reveal wound-ing-induced agarwood formation mechanism in two types of Aquilaria sinensis (Lour.) Spreng. Industrial Crops and Products. 2022;178:114603.

* View Article

* Google Scholar

11. 11. Liu X, Wang Y, Wang H, Wang L, Mei W, Dai H, et al. GC-MS analysis of chemical constituents of volatile oil from four kinds of agarwood tree leaves. Chinese Journal of Tropical Crops. 2022;43(1):196.

* View Article

* Google Scholar

12. 12. Wang YG, Wang J, Yang JL, Cai CH, Gai CJ, Mei WL. Bud grafting for propagation of Aquilaria sinensis ‘Reke2’. Chin J Trop Agri. 2020;40:79–88.

* View Article

* Google Scholar

13. 13. Yu M, Liu Y, Feng J, Chen D, Yang Y, Liu P, et al. Remarkable Phytochemical Characteristics of Chi-Nan Agarwood Induced from New-Found Chi-Nan Germplasm of Aquilaria sinensis Compared with Ordinary Agarwood. Int. J. Anal. Chem. 2021;33:533–542.

* View Article

* Google Scholar

14. 14. Liu JN, Whang LM, Zeng LM, Liu DQ. Agarwood planting promotes rural revitalization in Hainan Province. J Agric Catastrophol. 2021;11(1):143–4.

* View Article

* Google Scholar

15. 15. Liu XJ, Xu DP. Characteristics of resource distribution, industry status and development proposal of precious tree species in Guangdong. Guangdong Agricultural Sciences. 2021;48:57–65.

* View Article

* Google Scholar

16. 16. Fan HY, Huang Y, Deng QH, Song SQ. Agarwood family. Life World. 2021;380:12–7.

* View Article

* Google Scholar

17. 17. Zhu Y, Dumroese RK, Pinto JR, Li GL, Liu Y. Fall fertilization enhanced nitrogen storage and translocation in Larix olgensis seedlings. New Forests. 2013;44(4):849–61.

* View Article

* Google Scholar

18. 18. Kai YG. Industrialization of organic fertilizer and the sustainable agriculture development in China. Chinese Agricultural Science Bulletin. 2004.

* View Article

* Google Scholar

19. 19. Ping L, U NH, Bo L. Requirement in plant nutrition and fertilizer use of organic agriculture. Review of China Agricultural Science and Technology. 2004.

* View Article

* Google Scholar

20. 20. Watimena SF. Effect of organic and inorganic fertilizer application on soybeans growth and yield (Glycine max L. Merrill). Journal of Science and Science Education. 2023;4(1).

* View Article

* Google Scholar

21. 21. Singh A, Prasad VM, Bahadur V, Topno SE. Effect of organic and inorganic fertilizer on growth, yield and quality traits of cucumber (Cucumis sativus) under Prayagraj Agro-climatic condition AkiNik Publications, 2021;7.

22. 22. Mondal AB, Azad AK, Ahmed MB, Mannan A, Hossain M, Eaton TE. Influence of organic and inorganic fertilizers on floating bed cultivation of okra and cucumber during summer season in southern part of bangladesh. American Journal of Botany. 2022;5(13):005–13.

* View Article

* Google Scholar

23. 23. Amfo B, Ali EB. Beyond adoption: the interaction between organic and inorganic fertilizer application, and vegetable productivity in ghana. Renewable Agriculture and Food Systems. 2021;15(1):1–17.

* View Article

* Google Scholar

24. 24. Sun G. Experimental study on the correlation between different fertilization levels and the growth of young Cinnamomum camphora forest. Journal of Green Science and Technology. 2019;13(3):3.

* View Article

* Google Scholar

25. 25. Ji Y. Effects of Fertilization on Soil Fertility and Physiological Activity of Larix gmelinii Plantation, Northeast Forestry University, China; 2002.

26. 26. Tan C, Zhou JQ, Wang LH, Yin T, Tang CH, Liang X. Comparative experiment of different eucalyptus compound fertilizer base fertilizers. Forestry science and technology development. 2014.

* View Article

* Google Scholar

27. 27. Pang SJ, Zhang P, Yang BG, Liu SL, Feng CL. Effects of suitable ratio of biological fertilizer and chemical fertilizer on growth of Aquilaria sinensis and soil nutrients. Soil Fertil Sci. 2020;3:6.

* View Article

* Google Scholar

28. 28. Arunakumara K, Walpola B, Karunatunga C. Assessment of the early growth of agarwood (Aquilaria crassna) seedlings under different sources of nutrients. J For Sci. 2022;68(1):1–7.

* View Article

* Google Scholar

29. 29. Wang R, He Q, Ding XG, Li JY, Zhang FQ, Zhu BZ. Effects of exponential fertilization on seedling growth and photosynthesis of Aquilarias spp. Journal of Beijing Forestry University. 2011;33(7):7.

* View Article

* Google Scholar

30. 30. Wen JD. Comparative study on the physical and chemical properties of eucalyptus plantation soil in Guangxi Karst and red soil area. Ecology and Environmental Sciences. 2013.

* View Article

* Google Scholar

31. 31. Yang MW. Study on rapid determination of chlorophyll content of leaves. Chin J Spectrosc Lab. 2002;19:478–81.

* View Article

* Google Scholar

32. 32. Cai YP. Plant Physiology Experiment Instruction; China Agricultural University Press: Beijing, China, 2014. p. 166–70.

33. 33. Hao ZB, Cang J, Xu Z. Plant Physiology Experiment; Harbin Institute of Technology Press: Harbin, China; 2004.

34. 34. Li ZF, Wu XD. Experimental Design Scheme for the Effect of Drought Stress on Content of Malondialdehyde of Indoor Ornamental Plants. Tianjin Agric Sci. 2016;22:3.

* View Article

* Google Scholar

35. 35. Zhang ZA, Chen ZY. Experimental Techniques in Plant Physiology; Liaoning Science and Technology Press: Shenyang, China; 2008.

36. 36. Masayuki Y, Toshihiko H. A canopy photosynthesis model for the dynamics of size structure and self-thinning in plant populations. Annals of Botany. 1992;4:305–16.

* View Article

* Google Scholar

37. 37. Mazeh M, Almadi L, Paoletti A, Cinosi N, Daher E, Tucci M. Use of an Organic Fertilizer Also Having a Biostimulant Action to Promote the Growth of Young Olive Trees. 2021.

38. 38. Dai LL, Shi FQ, Liu YY, Yuan LZ, Xu YM. Topdressing experiment of forest plantation of Aquilaria yunnanensis. Journal of Shandong Forestry Science and Technology. 2017;47(3):3.

* View Article

* Google Scholar

39. 39. Feng HY, Li X, Xia SZ. Effects of different fertilization on the growth of Corylus heterophylla seedlings. Forest By-Product and Speciality in China. 2023;1(1):15–7.

* View Article

* Google Scholar

40. 40. Xing BW, Teng FD, Shi QL, Li MJ. Effects of formula fertilization on leaf nutrient dynamics and yield of sapindus mukorossi gaertn. Journal of Nanjing Forestry University (Natural Sciences Edition). 2018.

* View Article

* Google Scholar

41. 41. Xiao D, Liu X, Yang R, Tan Y, Wang K. Nitrogen input through chemical and biological pathway affects arbuscular mycorrhizal fungal communities in forage grass cultivation in southwest China. Applied Soil Ecology. 2019;150:103454.

* View Article

* Google Scholar

42. 42. Hao TW, Nie J, Yang WJ, Zhang ZY, Kang HW, Xiao YL. Effects of machine sowing depth and amounts of seeds and fertilizer on lodging and yield of Tartary buckwheat. Acta Prataculturae Sinica. 2020;29(12):61–72.

* View Article

* Google Scholar

43. 43. Otieno PC, Nyalala S, Wolukau J. Postharvest quality and safety of potted greenhouse tomato grown on forest soil-biosolids substrate, blended with NPK fertilizer. NASS Journal of Agricultural Sciences. 2020;2(2):13.

* View Article

* Google Scholar

44. 44. Shi YW, Wang YL, Wen BL. Effects of water stress on soluble protein, soluble sugar and proline content in Tamarix hispida. Journal of Xinjiang Agricultural University. 2007.

* View Article

* Google Scholar

45. 45. Watanabe S, Kojima K, Ide Y, et al. Effects of saline and osmotic stress on proline and sugar accumulation in Populus euphratica in vitro. Plant Cell, Tissue and Organ Culture. 2000;63(3):199–206.

* View Article

* Google Scholar

46. 46. Li Y, Chen ZL, Wang J. Effects of salt stress on growth and concentration of osmoregulatory substances of Suaeda heteropptera Kitagawa. Chinese Journal of Ecology. 2011;30(01):72–6.

* View Article

* Google Scholar

47. 47. Zhang L, Jia H, Rui P. Physiological performance of three calcifying green macroalgae Halimeda species in response to altered seawater temperatures. Acta Oceanologica Sinica. 2020;2(2):89–100.

* View Article

* Google Scholar

48. 48. Kabuto H, Hasuike S, Minagawa N, Shishibori T. Effects of bisphenol A on the metabolisms of active oxygen species in mouse tissues. Environ Res. 2003;93(1):31–5. pmid:12865045

* View Article

* PubMed/NCBI

* Google Scholar

49. 49. Wan S, Ling Q, Yu T. Effect of water stress on POD SOD activity and iso-POD SOD in Chinese chestnut seedlings leaves. Beijing Agricultural College. 1997;12:20–5.

* View Article

* Google Scholar

Citation: Huang J, Fu Y, Yu Z, Li X, Zheng S, Tang F, et al. (2025) Effects of organic-inorganic complex fertilizer on the growth and physiological characteristics of ‘Qi-Nan’ agarwood from Aquilaria sinensis (Lour.). PLoS ONE 20(4): e0320766. https://doi.org/10.1371/journal.pone.0320766

About the Authors:

Jingyue Huang

Roles: Investigation, Software, Writing – original draft

Affiliations: Guangxi Collegesand Universities Key Laboratory for Cultivation and Utilization of Subtropical Forest Plantation, College of Forestry, GuangxiUniversity, Nanning 530004, China, Guangxi Gaofeng State Owned Forest Farm, Nanning, China

Yunlin Fu

Roles: Funding acquisition, Supervision

Affiliation: Guangxi Collegesand Universities Key Laboratory for Cultivation and Utilization of Subtropical Forest Plantation, College of Forestry, GuangxiUniversity, Nanning 530004, China

Zhu Yu

Roles: Investigation, Software, Writing – original draft

Affiliation: Guangxi Forest Inventory & Planning Institute, Nanning, China

Xueting Li

Roles: Investigation

Affiliation: Guangxi Collegesand Universities Key Laboratory for Cultivation and Utilization of Subtropical Forest Plantation, College of Forestry, GuangxiUniversity, Nanning 530004, China

Siyu Zheng

Roles: Investigation

Affiliation: Guangxi Collegesand Universities Key Laboratory for Cultivation and Utilization of Subtropical Forest Plantation, College of Forestry, GuangxiUniversity, Nanning 530004, China

Fenyong Tang

Roles: Investigation

Affiliation: Guangxi Forest Inventory & Planning Institute, Nanning, China

Penglian Wei

Roles: Conceptualization, Writing – review & editing

E-mail: [email protected]

Affiliation: Guangxi Collegesand Universities Key Laboratory for Cultivation and Utilization of Subtropical Forest Plantation, College of Forestry, GuangxiUniversity, Nanning 530004, China

ORICD: https://orcid.org/0000-0002-9028-1824

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References

1. Ng LT. A review on agar (gaharu) producing Aquilaria species. J Tropic Forest Prod. 1997;2:272–85.

2. Barden A, Anak NA, Mulliken T, Song M. Heart of the matter: agarwood use and trade and CITES implementation for Aquilaria malaccensis. Traffic International. 2000;39:937–50.

3. Persoon GA, van Beek HH. Growing ‘the wood of the gods’: Agarwood production in southeast Asia. Smallholder Tree Growers and Rural Development Services. 2008;2:245–62.

4. Snelder D, Rodel DL. Smallholder Tree Growing for Rural Development and Environmental Services: Lessons from Asia. Springer Science & Business Media: Berlin/Heidelberg, Germany; 2008.

5. Yamagata E, Yoneda K. Pharmacognostical studies on the crude drug of agarwood (VI): On “Kanankoh”. Shoyakugaku Zasshi. 1987;41:142–6.

6. Nakanishi T, Inada A, Nishi M, Yamagata E, Yoneda K. A new and a known derivatives of 2-(2-phenylethyl) chromone from a kind of agarwood (“Kanankoh”, in Japanese) originating from Aquilaria agallocha. Journal of Natural Products. 1986;49(6):1106–8.

7. Azren PD, Lee SY, Emang D, Mohamed R. History and perspectives of induction technology for agarwood production from cultivated Aquilaria in Asia: A review. Journal of Forestry Research. 2019;30(1):1–11.

8. Naef R. The volatile and semi-volatile constituents of agarwood, the infected heartwood of Aquilaria species: A review. Flavour and Fragrance Journal. 2011;26(1):73–87.

9. Liu J, Yang J, Jiang C, Zhou J, Zhao Y, Huang L. Volatile organic compound and endogenous phytohormone characteristics during callus browning in Aquilaria sinensis. Ind Crops Prod. 2021;168:113605. Convention on Internal Trade in Endangered Species of Wild Fauna and Flora.

10. Zhang P, Li X, Cui Z, Xu D. Morphological, physiological, biochemical and molecular analyses reveal wound-ing-induced agarwood formation mechanism in two types of Aquilaria sinensis (Lour.) Spreng. Industrial Crops and Products. 2022;178:114603.

11. Liu X, Wang Y, Wang H, Wang L, Mei W, Dai H, et al. GC-MS analysis of chemical constituents of volatile oil from four kinds of agarwood tree leaves. Chinese Journal of Tropical Crops. 2022;43(1):196.

12. Wang YG, Wang J, Yang JL, Cai CH, Gai CJ, Mei WL. Bud grafting for propagation of Aquilaria sinensis ‘Reke2’. Chin J Trop Agri. 2020;40:79–88.

13. Yu M, Liu Y, Feng J, Chen D, Yang Y, Liu P, et al. Remarkable Phytochemical Characteristics of Chi-Nan Agarwood Induced from New-Found Chi-Nan Germplasm of Aquilaria sinensis Compared with Ordinary Agarwood. Int. J. Anal. Chem. 2021;33:533–542.

14. Liu JN, Whang LM, Zeng LM, Liu DQ. Agarwood planting promotes rural revitalization in Hainan Province. J Agric Catastrophol. 2021;11(1):143–4.

15. Liu XJ, Xu DP. Characteristics of resource distribution, industry status and development proposal of precious tree species in Guangdong. Guangdong Agricultural Sciences. 2021;48:57–65.

16. Fan HY, Huang Y, Deng QH, Song SQ. Agarwood family. Life World. 2021;380:12–7.

17. Zhu Y, Dumroese RK, Pinto JR, Li GL, Liu Y. Fall fertilization enhanced nitrogen storage and translocation in Larix olgensis seedlings. New Forests. 2013;44(4):849–61.

18. Kai YG. Industrialization of organic fertilizer and the sustainable agriculture development in China. Chinese Agricultural Science Bulletin. 2004.

19. Ping L, U NH, Bo L. Requirement in plant nutrition and fertilizer use of organic agriculture. Review of China Agricultural Science and Technology. 2004.

20. Watimena SF. Effect of organic and inorganic fertilizer application on soybeans growth and yield (Glycine max L. Merrill). Journal of Science and Science Education. 2023;4(1).

21. Singh A, Prasad VM, Bahadur V, Topno SE. Effect of organic and inorganic fertilizer on growth, yield and quality traits of cucumber (Cucumis sativus) under Prayagraj Agro-climatic condition AkiNik Publications, 2021;7.

22. Mondal AB, Azad AK, Ahmed MB, Mannan A, Hossain M, Eaton TE. Influence of organic and inorganic fertilizers on floating bed cultivation of okra and cucumber during summer season in southern part of bangladesh. American Journal of Botany. 2022;5(13):005–13.

23. Amfo B, Ali EB. Beyond adoption: the interaction between organic and inorganic fertilizer application, and vegetable productivity in ghana. Renewable Agriculture and Food Systems. 2021;15(1):1–17.

24. Sun G. Experimental study on the correlation between different fertilization levels and the growth of young Cinnamomum camphora forest. Journal of Green Science and Technology. 2019;13(3):3.

25. Ji Y. Effects of Fertilization on Soil Fertility and Physiological Activity of Larix gmelinii Plantation, Northeast Forestry University, China; 2002.

26. Tan C, Zhou JQ, Wang LH, Yin T, Tang CH, Liang X. Comparative experiment of different eucalyptus compound fertilizer base fertilizers. Forestry science and technology development. 2014.

27. Pang SJ, Zhang P, Yang BG, Liu SL, Feng CL. Effects of suitable ratio of biological fertilizer and chemical fertilizer on growth of Aquilaria sinensis and soil nutrients. Soil Fertil Sci. 2020;3:6.

28. Arunakumara K, Walpola B, Karunatunga C. Assessment of the early growth of agarwood (Aquilaria crassna) seedlings under different sources of nutrients. J For Sci. 2022;68(1):1–7.

29. Wang R, He Q, Ding XG, Li JY, Zhang FQ, Zhu BZ. Effects of exponential fertilization on seedling growth and photosynthesis of Aquilarias spp. Journal of Beijing Forestry University. 2011;33(7):7.

30. Wen JD. Comparative study on the physical and chemical properties of eucalyptus plantation soil in Guangxi Karst and red soil area. Ecology and Environmental Sciences. 2013.

31. Yang MW. Study on rapid determination of chlorophyll content of leaves. Chin J Spectrosc Lab. 2002;19:478–81.

32. Cai YP. Plant Physiology Experiment Instruction; China Agricultural University Press: Beijing, China, 2014. p. 166–70.

33. Hao ZB, Cang J, Xu Z. Plant Physiology Experiment; Harbin Institute of Technology Press: Harbin, China; 2004.

34. Li ZF, Wu XD. Experimental Design Scheme for the Effect of Drought Stress on Content of Malondialdehyde of Indoor Ornamental Plants. Tianjin Agric Sci. 2016;22:3.

35. Zhang ZA, Chen ZY. Experimental Techniques in Plant Physiology; Liaoning Science and Technology Press: Shenyang, China; 2008.

36. Masayuki Y, Toshihiko H. A canopy photosynthesis model for the dynamics of size structure and self-thinning in plant populations. Annals of Botany. 1992;4:305–16.

37. Mazeh M, Almadi L, Paoletti A, Cinosi N, Daher E, Tucci M. Use of an Organic Fertilizer Also Having a Biostimulant Action to Promote the Growth of Young Olive Trees. 2021.

38. Dai LL, Shi FQ, Liu YY, Yuan LZ, Xu YM. Topdressing experiment of forest plantation of Aquilaria yunnanensis. Journal of Shandong Forestry Science and Technology. 2017;47(3):3.

39. Feng HY, Li X, Xia SZ. Effects of different fertilization on the growth of Corylus heterophylla seedlings. Forest By-Product and Speciality in China. 2023;1(1):15–7.

40. Xing BW, Teng FD, Shi QL, Li MJ. Effects of formula fertilization on leaf nutrient dynamics and yield of sapindus mukorossi gaertn. Journal of Nanjing Forestry University (Natural Sciences Edition). 2018.

41. Xiao D, Liu X, Yang R, Tan Y, Wang K. Nitrogen input through chemical and biological pathway affects arbuscular mycorrhizal fungal communities in forage grass cultivation in southwest China. Applied Soil Ecology. 2019;150:103454.

42. Hao TW, Nie J, Yang WJ, Zhang ZY, Kang HW, Xiao YL. Effects of machine sowing depth and amounts of seeds and fertilizer on lodging and yield of Tartary buckwheat. Acta Prataculturae Sinica. 2020;29(12):61–72.

43. Otieno PC, Nyalala S, Wolukau J. Postharvest quality and safety of potted greenhouse tomato grown on forest soil-biosolids substrate, blended with NPK fertilizer. NASS Journal of Agricultural Sciences. 2020;2(2):13.

44. Shi YW, Wang YL, Wen BL. Effects of water stress on soluble protein, soluble sugar and proline content in Tamarix hispida. Journal of Xinjiang Agricultural University. 2007.

45. Watanabe S, Kojima K, Ide Y, et al. Effects of saline and osmotic stress on proline and sugar accumulation in Populus euphratica in vitro. Plant Cell, Tissue and Organ Culture. 2000;63(3):199–206.

46. Li Y, Chen ZL, Wang J. Effects of salt stress on growth and concentration of osmoregulatory substances of Suaeda heteropptera Kitagawa. Chinese Journal of Ecology. 2011;30(01):72–6.

47. Zhang L, Jia H, Rui P. Physiological performance of three calcifying green macroalgae Halimeda species in response to altered seawater temperatures. Acta Oceanologica Sinica. 2020;2(2):89–100.

48. Kabuto H, Hasuike S, Minagawa N, Shishibori T. Effects of bisphenol A on the metabolisms of active oxygen species in mouse tissues. Environ Res. 2003;93(1):31–5. pmid:12865045

49. Wan S, Ling Q, Yu T. Effect of water stress on POD SOD activity and iso-POD SOD in Chinese chestnut seedlings leaves. Beijing Agricultural College. 1997;12:20–5.