A method of citrus cultivation

Abstract

The present application provides a citrus planting method, wherein a diluent of concentrated Rhodopseudomonas spheroides metabolites is applied in a root irrigation manner during the fruit expansion period of the citrus fruit trees. The present application finds that the concentrated Rhodopseudomonas spheroides metabolites can help delay the ripening of the citrus fruits and improve the fruit quality. When the fruits of red beauties are irrigated in a specific root irrigation manner, the soluble solid content in the fruits of the red beauties can be increased, and the fruits can be made to ripen later to accumulate more nutritional ingredients. Meanwhile, it is accidentally found that the copper content in the soil for planting the red beauties is significantly reduced, and the zinc content is significantly increased, which proves that the citrus planting method provided by the present application can not only obtain a kind of citrus fruit with higher nutritional value and flavor value, but also can reduce the copper toxicity caused by the acidic soil environment in the plant.

Description

Technical Field

[0001] This invention relates to the field of microbial application technology, specifically to a method for citrus cultivation. Background Technology

[0002] Rhodobacter sphaeroides, belonging to the phylum Violet Nonsulfur Bacteria, is a Gram-negative prokaryotic photosynthetic bacterium (PSB). This species possesses a photosynthetic system, enabling it to convert light energy into chemical energy through photosynthesis under anaerobic conditions, utilizing environmental organic compounds and ammonia as hydrogen donors and carbon sources. Rhodobacter sphaeroides exhibits remarkable environmental adaptability, surviving in aerobic, anaerobic, and dark conditions. Its metabolism synthesizes various bioactive substances, including carotenoids, coenzyme Q10, superoxide dismutase (SOD), 5-aminolevulinic acid (ALA), and hydrogen. These metabolites are used not only as microbial pesticides in agriculture to promote plant growth and enhance disease resistance, but also show potential in environmental protection, such as degrading pesticide residues, organic wastewater, and toxic substances like polycyclic aromatic hydrocarbons (PAHs). Therefore, Rhodopsycetes have broad application prospects in agriculture and environmental protection, and are considered to have significant potential for industrial development.

[0003] The application of specific, highly efficient photosynthetic bacteria inoculants has significant positive effects on plant growth, such as promoting increased plant height and stem diameter, as well as improving fresh and dry weight. Studies have found that photosynthetic bacteria can increase chlorophyll content and root nodule number in soybean plants, thereby promoting nutrient absorption, increasing yield, and improving soybean quality. Furthermore, photosynthetic bacteria treatment can improve rice seed germination rate and promote rice growth. Similarly, photosynthetic bacteria have the potential to induce plant resistance and can be used as surface application agents for plant protection, providing diverse plant-microbe interaction patterns. Further research has shown that applying diluted Rhodopseudomonas aeruginosa inoculants to the leaves of tomatoes and strawberries can form dominant microbial communities on the leaves, positively impacting plant growth, flowering, yield increase, disease resistance, and quality improvement. This treatment significantly reduces the incidence of gray mold and the risk of low-temperature damage, thereby improving overall crop health and yield.

[0004] In conclusion, the metabolites of Rhodopseudomonas spp. show significant application potential in the agricultural field, especially in improving fruit flavor and enhancing fruit quality. Summary of the Invention

[0005] This invention provides a method for cultivating citrus. This application discovers that concentrated metabolites of *Rhodophyta globulina* can help delay the ripening of citrus fruits and improve fruit quality. When applied to the *Red Beauty* citrus plant via root irrigation, it helps increase the soluble solids content in the fruit and promotes delayed ripening to accumulate more nutrients. Simultaneously, it was unexpectedly found that the copper content in the soil where *Red Beauty* is planted is significantly reduced, while the zinc content is significantly increased. This demonstrates that the citrus cultivation method provided in this application, through root irrigation with different concentrations of *Rhodophyta globulina* metabolites, can significantly reduce the copper content in the roots of *Red Beauty* citrus, thereby reducing copper toxicity caused by acidic soil environments.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for citrus cultivation in which a diluted agent of concentrated metabolites of Rhodotorula glutinis is applied by root irrigation during the fruit enlargement period of citrus trees.

[0008] Preferably, the concentrated metabolites of the *Rhodotorula globulus* comprise: 16.97 ng / mL of hexadecanoic acid, 11.55 ng / mL of 2-pyridone, 8.64 ng / mL of stearic acid, 4.52 ng / mL of 3-hydroxypropionic acid, 3.27 ng / mL of phosphate, 3.24 ng / mL of lactic acid, 2.25 ng / mL of citric acid, 1.21 ng / mL of 2-ketoisovaleric acid, 1.15 ng / mL of 1,5-anhydrous glucose, 0.99 ng / mL of hydroxylamine, 1.04 ng / mL of uracil, 0.9 ng / mL of 4-aminobutyric acid, 0.97 ng / mL of L-citrulline, 0.88 ng / mL of β-alanine, and 0.79 ng / mL of acetaminophen.

[0009] Preferably, the concentrated metabolites of the *Rhodotorula globulus* include the following hormones: 0.13 ng / mL indoleacetamide, 4.73 ng / mL salicylic acid, 0.10 ng / mL gibberellin A3, 10.58 ng / mL indolecarboxylic acid, 9.17 ng / mL indole-3-acetic acid, 0.53 ng / mL abscisic acid, 79.33 ng / mL indolepropionic acid, 0.26 ng / mL jasmonic acid, 0.05 ng / mL gibberellin A7, 0.17 ng / mL gibberellin A4, 0.32 ng / mL dihydrojasmonic acid, and 0.22 ng / mL jasmonic acid-isoleucine.

[0010] Preferably, the Rhodotorula globulus is Rhodotorula globulus RD2-3, deposited at the China Center for Type Culture Collection, with accession number CCTCC No: M 20211528.

[0011] Preferably, the diluent is diluted by a factor of 50 to 150.

[0012] Preferably, the citrus tree variety is Red Beauty.

[0013] Preferably, the citrus trees are cultivated by planting them at equal intervals of 4-5m between rows and 4-5m between plants.

[0014] Preferably, the diluent for the concentrated metabolites of Rhodotorula globulus is applied in three separate applications, with equal time intervals between each application.

[0015] Preferably, the root irrigation method is as follows: dig down more than 0.2m perpendicular to the ground to expose the main root of the citrus tree, and then apply a diluent of concentrated metabolites of Rhodopseudomonas spp.

[0016] Preferably, when applying a diluent for concentrated metabolites of Rhodopseudomonas spp. in a single application, half of the total amount is applied within 20 cm of the main root, and the remaining half is applied beyond 20 cm of the main root.

[0017] Therefore, the present invention has the following beneficial effects:

[0018] (1) The citrus planting method provided by the present invention can not only obtain a citrus fruit with higher nutritional and flavor value, but also reduce copper toxicity caused by acidic soil environment in plants.

[0019] (2) This invention directly improves the quality of citrus fruit by applying the metabolites of Rhodotorula globulus, and indirectly improves the quality of citrus fruit by delaying the ripening of citrus fruit by applying the metabolites of Rhodotorula globulus.

[0020] (3) This invention delays the ripening of citrus fruits by applying a concentrated metabolite diluent to Rhodotorula glutinis during the fruit enlargement period, thereby extending the time for sugar accumulation in the fruit and having an indirect positive impact on improving fruit quality.

[0021] (4) This invention replaces microbial fertilizer by adding concentrated metabolites of Rhodotorula glutinis to the roots, focusing on using the secondary metabolites of microorganisms to affect plants, regulate the expression of genes related to citrus fruit growth, and thus promote citrus fruit growth.

[0022] (5) This invention improves fruit quality by regulating plant hormone signal transduction and metabolic processes through the combined action of 4-aminobutyric acid and auxin substances in the metabolites of Rhodotorula globulus. Attached Figure Description

[0023] Figure 1 Root irrigation with Rhodotorula glutinis metabolites affects the ripening of 'Red Beauty' fruit;

[0024] Figure 2 The effect of Rhodopseudomonas globosum metabolites on the soluble solids content of 'Red Beauty' fruit was investigated. Different lowercase letters indicate significant differences (P < 0.05).

[0025] Figure 3 The effect of root irrigation with Rhodotorula glutinis metabolites on the sugar and acid content of 'Red Beauty' fruit was investigated. Different lowercase letters indicate significant differences (P < 0.05).

[0026] Figure 4 The effect of root irrigation with Rhododendron globosum metabolites on nutrient content in the leaves and roots of 'Red Beauty' succulents was investigated. Different lowercase letters indicate significant differences (P < 0.05).

[0027] Figure 5 The correlation between fruit quality and nutrient content of Red Beauty fruit under root irrigation treatment with Rhododendron simsii metabolites;

[0028] Figure 6 The results show the trace element determination of citrus roots under root irrigation treatment with Rhodotorula glutinis metabolites. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0030] In this section, all statistical analyses were performed using SPSS, Prism 9 (USA), and Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). Correlation analysis was conducted using the online website Chiplot (https: / / www.chiplot.online / ).

[0031]

Example

[0032] ①Preparation methods of Rhodopseudomonas aeruginosa metabolites

[0033] The Rhodocytogenes RD2-3 strain described in this section was purchased from Green Ao Environmental Technology (Shanghai) Co., Ltd. This strain has been disclosed in patent CN114317357B.

[0034] Aseptically scraping the bacterial colony of *Rhodopseudomonas RD2-3*, inoculating it into 20 mL of *Rhodopseudomonas RD2-3* fermentation medium (commercially available conventional *Rhodopseudomonas RD2-3* medium can be used), and incubating at 35°C with shaking for 48 hours until the medium turns light red; then transferring it to 100 mL of *Rhodopseudomonas RD2-3* fermentation medium, and incubating at 35°C with shaking for 32 hours until it turns red, then transferring it to 400 mL of *Rhodopseudomonas RD2-3* fermentation medium, and incubating at 35°C with shaking for 32 hours to obtain the deep red fermentation broth, is the *Rhodopseudomonas RD2-3* metabolite of this invention. The bacterial concentration of this *Rhodopseudomonas RD2-3* metabolite was determined to be approximately 10⁻⁶. 8 ~10 9 CFU / mL.

[0035] ② Determination of the components of metabolites of Rhodopseudomonas aeruginosa

[0036] Determination of concentrated metabolites and hormone content in Rhodopseudomonas aeruginosa: Concentrated metabolites of Rhodopseudomonas aeruginosa were directly analyzed using non-target metabolomics and hormone-target metabolomics, with mobile phase parameters controlled by an Agilent 1290 ultra-high performance liquid chromatography (UPLC) system. The chromatographic column used was a Waters UPLC BEH Amide column (1.7 μm * 2.1 mm * 100 mm).

[0037] Table 1 shows the composition of the metabolites of *Rhodotorula globulus*. Analysis reveals that the metabolites are mainly composed of organic acids and amino acids, with organic acids making up a particularly significant proportion. Of particular note is the detection of trace amounts of 4-aminobutyric acid (GABA) in these metabolites. GABA acts as a signaling molecule, promoting chlorophyll synthesis, and is also a precursor in the biosynthesis of the plant hormone abscisic acid (ABA). Spraying GABA can induce an increase in the levels of endogenous GABA and plant hormones (such as gibberellin and indoleacetic acid), participating in metabolic regulation during fruit ripening and thus improving fruit quality to some extent.

[0038] Table 2 shows the hormone components and their corresponding contents in the metabolites of *Rhodotorula globulus*. The results indicate that these metabolites contain small amounts of plant hormones, mainly auxins, specifically indoleacetic acid (IAA), indole-3-acetic acid (IAA), and indolepropionic acid (IPA). In addition, salicylic acid was also detected at a concentration of 4.73 ng / mL. Based on the information in Table 2, it can be inferred that 4-aminobutyric acid (GABA) and auxins in the *Rhodotorula globulus* metabolites may work together to improve fruit quality by regulating plant hormone signal transduction and metabolic processes.

[0039] Table 1. Composition of metabolites from Rhodotorula glutinis in non-targeted metabolic assays

[0040] palmitic acid 16.97 hexadecanoic acid 2-hydroxypyridine 11.55 2-Pyridone stearic acid 8.64 stearic acid 3-Hydroxypropionic acid 1 4.52 3-Hydroxypropionic acid phosphate 3.27 phosphate lactic acid 3.24 lactic acid citric acid 2.25 Citric acid 2-keto-isovaleric acid 1 1.21 2-Ketoisovalerate 1,5-Anhydroglucitol 1.15 1,5-Anhydrous Glucoside hydroxylamine 0.99 hydroxylamine uracil 1.04 Uracil 4-aminobutyric acid 3 0.9 4-Aminobutyric acid citrulline 2 0.97 L-citrulline beta-Alanine 2 0.88 β-alanine 4-Acetamidobutyric acid 2 0.79 Acetaminobutyric acid

[0041] Table 2 Hormone content of metabolites from Rhodotorula glutinis

[0042] 3-Indoleacetamide 0.13 Indoleacetamide Salicylic Acid 4.73 salicylic acid Gibberellic A3 0.10 Gibberellin A3 3-Indolecarboxylic acid 10.58 Indolecarboxylic acid Indole-3-Acetic Acid 9.17 Indole-3-acetic acid Abscisic Acid 0.53 abscisic acid 3-Indolepropionic acid 79.33 Indolepropionic acid Jasmonic Acid 0.26 Jasmonic acid Gibberellin A7 0.05 Gibberellin A7 Gibberellin A4 0.17 Gibberellin A4 Dihydrojasmonic acid 0.32 Dihydrojasmonic acid Jasmonic acid-Isoleucine 0.22 Jasmonic acid-isoleucine

[0043] ③ Red Beauty Planting

[0044] Greenhouse setup: Evenly growing 'Hong Mei Ren' 'Citrus reticulata' fruit trees were selected and planted in the greenhouse planting area of ​​Zhejiang Yunzeying Agricultural Technology Development Co., Ltd., Kecheng District, Quzhou City, covering an area of ​​approximately 667 square meters. 2 The row spacing is 4 meters and the plant spacing is 4 meters.

[0045] From July 16, 2024 to September 17, 2024, during the fruit enlargement period of Red Beauty berry, some Red Beauty berry trees in the greenhouse were irrigated with a concentrated metabolite diluent of Rhodotorula glutinis (diluted at 30, 50, 100, and 150 times) every month (the first application was on July 16, 2024, the second application was on August 16, 2024, and the third application was on September 16, 2024). Specific treatments can be found in Table 3.

[0046] Root irrigation treatment method: Dig vertically into the ground at least 0.2m deep to expose the main root of the citrus tree, and then apply a diluted solution of concentrated metabolites of Rhodopseudomonas aeruginosa. When applying the diluted solution of concentrated metabolites of Rhodopseudomonas aeruginosa in a single application, apply half of the total amount within 20cm of the main root and the other half at a distance of more than 20cm from the main root.

[0047] Table 3 Experimental Design Processing

[0048]

[0049] [Performance Testing Methods]

[0050] The testing methods in this section are as follows:

[0051] ① In the above experimental section, five trees with similar growth were selected for each treatment for investigation. Each tree was labeled. For the Red Beauty citrus, samples were collected at the early color-changing stage (October 17), mid-color-changing stage (October 30), and ripening stage (November 17). For the grapefruit, only samples were collected at the ripening stage (November 30). The effects of Rhodophyta globosum on citrus quality were analyzed. The survey data mainly included the nutrient status of Red Beauty leaves and roots, and the fruit quality indicators of Red Beauty and grapefruit.

[0052] ② Determination of soluble solids content in citrus fruits: The soluble solids content of the fruits was determined using a PR-101 digital refractometer. Five biological replicates were set up for each experiment, with 10 fruits in each replicate. The samples were mixed by juicing the fruit pulp.

[0053] ③ Determination of titratable acid content in citrus fruit: Weigh 5g of citrus pulp sample and add 10mL of distilled water. Homogenize and extract in an 80℃ constant temperature water bath for 30min, shaking 3-4 times during the process. After cooling, centrifuge at 8000g for 10min and collect the supernatant. Titrate with 0.1mol / L NaOH standard solution until the solution turns slightly pink and does not fade within 30s, which is the endpoint. Record the amount of NaOH used, and calculate the titratable acid content based on the volume of NaOH solution consumed.

[0054] ④ Determination of total sugar content in citrus fruit: Weigh 0.1g of fresh sample, homogenize with 4mL of 80% ethanol solution, transfer to a 5mL centrifuge tube, incubate at 80℃ for 30min, centrifuge at 3000g for 5min, transfer the supernatant, and repeat the above steps 3 times. The supernatant is used for the determination of soluble total sugar. Take 50μL of the above supernatant and 250μL of 70% anthrone-sulfuric acid solution into a 2mL centrifuge tube, incubate at 95℃ for 10min (tightly cover to prevent moisture loss), cool naturally to room temperature, take 200μL into a 96-well plate, and measure the absorbance value A at a wavelength of 620nm.

[0055] ⑤ Determination of Nutrient Content in Citrus Leaves: The first nutrient leaf from a citrus fruit was dried at 105℃ for 30 minutes to kill the greenness, and then dried at 70℃ to a constant weight. The sample was weighed using a 1 / 1,000,000 balance and ground into powder using a TL 2010S high-throughput tissue grinder. 0.03g of the pulverized sample was placed in a digestion tube, and 6mL of a mixed acid (concentrated nitric acid:perchloric acid 4:1) was added. Then, a temperature gradient digestion was performed (60℃ for 1h, 120℃ for 1h, 150℃ for 1h, 190℃ to remove acid) until the perchloric acid began to decompose and white fumes appeared. The sample was then evaporated to dryness and cooled. 20mL of deionized water was added, and the volume was adjusted to 50mL. The sample was then filtered through a 0.45μm filter membrane, and the elemental content was determined by ICP-AES (inductively coupled plasma atomic emission spectrometry).

[0056] ⑥ Determination of Nutrient Content in Citrus Roots: Lateral roots of citrus trees were dug from the soil layer at a depth of 20-60 cm. Fine citrus roots were collected, dried at 105℃ for 30 min to kill the greenness, and then dried at 70℃ to a constant weight. The samples were weighed using a 1 / 1,000,000 balance and ground into powder using a TL2010S high-throughput tissue grinder. 0.03 g of the pulverized sample was placed in a digestion tube, and 6 mL of a mixed acid (concentrated nitric acid:perchloric acid 4:1) was added. A temperature gradient digestion was then performed (60℃ for 1 h, 120℃ for 1 h, 150℃ for 1 h, 190℃ to remove acid) until the perchloric acid began to decompose and white fumes appeared. The mixture was then evaporated to dryness and cooled. 20 mL of deionized water was added, and the volume was adjusted to 50 mL. The mixture was then filtered through a 0.45 μm filter membrane, and the elemental content was determined using ICP-AES (Inductively Coupled Plasma Atomic Emission Spectrometry).

[0057] ⑦ Determination of nitrogen content in citrus leaves and roots: Take 0.05g of the above-mentioned leaf and root samples, add 5ml of concentrated sulfuric acid, digest at 190℃ for 30min, add 5ml of 30% hydrogen peroxide, digest at 280℃ for 30min, take out after the digestion solution is completely clear, cool and make up to the 100ml mark, filter with a 0.45 filter membrane and determine the total nitrogen content using a flow cytometry automatic analyzer (AA3, SealCo, Germany).

[0058] [Performance Test Results]

[0059] 1. Effects of different dilution concentrations of Rhodotorula glutinis on the soluble solids content of Rhodotorula rubrum.

[0060] Color change was observed for all treatments, and the results are as follows: Figure 1 As shown; soluble solids tests were performed on all treatments, and the results are as follows. Figure 2 As shown in Table 4.

[0061] according to Figure 1 and Figure 2 As shown, the experimental groups were treated with concentrated metabolites of Rhodopseudomonas aeruginosa via root irrigation during the fruit enlargement period. The results showed that the fruits in all root irrigation treatment groups exhibited delayed ripening. Among them, the delayed color change was more pronounced in the Red Beauty root irrigation group at G30, G50, and G100.

[0062] Furthermore, compared to the untreated control group, the root irrigation treatments (G50, G100, and G150) on the Red Beauty mandarin orange trees significantly increased the soluble solids content of the fruit, by 19.3%, 23.9%, and 12.9%, respectively, compared to the control group. Among them, the treatment with root irrigation diluted 100 times (G100) showed the most significant effect on increasing the soluble solids content.

[0063] Considering both the delayed color change phenomenon and the increased content of soluble solids, the G50 and G100 groups showed the most significant effects on delaying the ripening of Red Beauty grapes and improving their quality and flavor.

[0064] Table 4. Effects of Rhodophyton floccosum metabolite treatment on the soluble solids content of Red Beauty fruit.

[0065]

[0066] 2. Effects of different treatments on the sugar and acid content of 'Red Beauty' grapes

[0067] Depend on Figure 3 As shown in Table 5, root irrigation with Rhodopseudomonas aeruginosa metabolites increased the total soluble sugar content in 'Red Beauty' berries when the dilution concentration exceeded 50 times, with the 100-fold dilution showing the best promoting effect. Furthermore, root irrigation treatment increased both sugar and titratable acid content in the fruit.

[0068] Table 5. Effects of root irrigation treatment with Rhodophyton floccosum metabolites on the sugar and acid content of Red Beauty fruit.

[0069]

[0070] 3. Effects of different treatments on nutrient content in the leaves and roots of 'Red Beauty' succulent

[0071] Figure 4 Table 6 shows the effects of root irrigation treatment with Rhodophyta globulus metabolites on the nutrient content of leaves and roots of 'Red Beauty' succulents. According to... Figure 4 As shown in Table 6, root irrigation with *Rhodotorula globulus* metabolites increased nitrogen content in leaves to varying degrees, leading to delayed fruit ripening, a phenomenon consistent with the trend of delayed fruit ripening. However, this treatment did not significantly affect phosphorus and potassium content in leaves. Compared to the control group (CK), root nitrogen content significantly increased only when *Rhodotorula globulus* metabolites were diluted 30-fold, while varying degrees of phosphorus content in roots were observed to decrease. Root irrigation had no significant effect on potassium content in roots.

[0072] Table 6. Effects of root irrigation treatment with Rhodophyton floccosum metabolites on nutrient content in leaves and roots of Rhodophyton floccosum var. rubrum.

[0073]

[0074] 4. Effects of different treatments on soil nutrient content

[0075] Table 7 shows that root irrigation with Rhodopseudomonas aeruginosa metabolites slightly lowered soil pH but had no significant effect on soil electrical conductivity. Regarding soil nutrients, only treatments with Rhodopseudomonas aeruginosa metabolites diluted 100-fold significantly increased ammonium nitrogen content, and all root irrigation treatments led to an increase in nitrate nitrogen content. Specifically, compared to the control group, the available potassium content in the G100 and G150 treatment groups was significantly reduced by 28.6% and 31.0%, respectively.

[0076] Table 7. Effects of root irrigation treatment with Rhodophyton floccosum metabolites on soil nutrient content.

[0077]

[0078] 5. Effects of different treatments on soil nutrient content

[0079] observe Figure 5 Correlation analysis was conducted to investigate the effects of root irrigation treatment with Rhodotorula glutinis metabolites on the fruit quality of Red Beauty jujube trees, and its correlation with tree nutrients and soil nutrient content. The results showed that under Rhodotorula glutinis metabolite root irrigation treatment, the fruit quality of Red Beauty jujube trees was significantly correlated with the phosphorus content in the roots and the available potassium content in the soil (p>0.65), while the correlation with other nutrient indicators was relatively low.

[0080] 6. Effects of different treatments on the content of trace metals in citrus roots

[0081] observe Figure 6 As shown in Table 8, applying different concentrations of Rhodopseudomonas aeruginosa metabolites through root irrigation can significantly reduce the copper content in the roots of Rhodops rubrum, thereby reducing copper toxicity caused by acidic soil environment in the plant; at the same time, it can increase zinc content and promote plant growth.

[0082] Table 8. Results of trace element determination of Rhodophytic metabolites in citrus roots under root irrigation treatment.

[0083]

Claims

1. A method for cultivating citrus, characterized in that, During the fruit enlargement period of citrus trees, apply a diluted agent of concentrated metabolites of Rhodotorula glutinis by root irrigation. The Rhodotorula globulus is Rhodotorula globulus RD2-3, deposited at the China Center for Type Culture Collection, accession number CCTCC No: M 20211528; the dilution factor of the diluent is 50-150 times; The root irrigation method is as follows: dig vertically downwards at least 0.2 m to expose the main root of the citrus tree, and then apply a diluent of concentrated metabolites of Rhodopseudomonas spp.; when applying the diluent of concentrated metabolites of Rhodopseudomonas spp. in a single application, apply 1 / 2 of the total amount within 20 cm of the main root and apply the remaining 1 / 2 at a distance of more than 20 cm from the main root. The concentrated metabolites of the aforementioned Rhodotorula globulus include: 16.97 ng / mL of hexadecanoic acid, 11.55 ng / mL of 2-pyridone, 8.64 ng / mL of stearic acid, 4.52 ng / mL of 3-hydroxypropionic acid, 3.27 ng / mL of phosphate, 3.24 ng / mL of lactic acid, 2.25 ng / mL of citric acid, 1.21 ng / mL of 2-ketoisovaleric acid, 1.15 ng / mL of 1,5-anhydrous glucose, 0.99 ng / mL of hydroxylamine, 1.04 ng / mL of uracil, 0.9 ng / mL of 4-aminobutyric acid, 0.97 ng / mL of L-citrulline, 0.88 ng / mL of β-alanine, and 0.79 ng / mL of acetaminophen. The concentrated metabolites of the *Rhodotorula globulus* include the following hormones: 0.13 ng / mL indoleacetamide, 4.73 ng / mL salicylic acid, 0.10 ng / mL gibberellin A3, 10.58 ng / mL indolecarboxylic acid, 9.17 ng / mL indole-3-acetic acid, 0.53 ng / mL abscisic acid, 79.33 ng / mL indolepropionic acid, 0.26 ng / mL jasmonic acid, 0.05 ng / mL gibberellin A7, 0.17 ng / mL gibberellin A4, 0.32 ng / mL dihydrojasmonic acid, and 0.22 ng / mL jasmonic acid-isoleucine.

2. The citrus planting method as described in claim 1, characterized in that, The citrus tree variety mentioned is Red Beauty.

3. The citrus planting method as described in claim 1, characterized in that, The citrus trees are cultivated by planting them at equal intervals of 4-5 m between rows and 4-5 m between plants.

4. The citrus planting method as described in claim 1, characterized in that, The diluent for the concentrated metabolites of the Rhodotorula globulus was applied in three separate applications, with equal time intervals between each application.

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