Application of Chlorella extracellular metabolites in promoting growth and resisting stress in higher plants

Abstract

The present invention cultivates Chlorella and prepares its extracellular metabolites, which are then used to promote plant growth and / or improve plant stress resistance. Chlorella extracellular metabolites promote lateral root elongation and cold resistance of Arabidopsis thaliana and Nicotiana benthamiana. Chlorella extracellular metabolites fermented at high cell density can promote the growth of rapeseed and vegetables such as tomatoes, cucumbers, Chinese cabbage, and lettuce when diluted 500 to 1000 times. Experiments also found that the yield of rapeseed treated with Chlorella extracellular metabolites was increased, and the vitamin C and protein content in the vegetables increased. This shows that Chlorella extracellular metabolites can not only enhance the cold resistance (stress resistance) of vegetables, but also increase the yield and nutritional value of leaf vegetables, providing a new technical research and development direction for the cultivation of high-quality organic vegetables. The present invention also provides a resource processing method for the supernatant after harvesting Chlorella culture and a preparation method for a natural plant stimulant.

Description

Technical Field

[0001] The present invention relates to the field of plant stimulants, and in particular to the application of extracellular metabolites of Chlorella vulgaris in promoting growth and resisting stress in higher plants. Background Art

[0002] Modern high-yield agricultural crop production relies primarily on chemical fertilizers. However, in recent years, the impact of chemical fertilizers on soil, water, air pollution, and food safety has received increasing attention. Biostimulants, as a promising fertilizer additive, can promote plant growth or improve crop stress resistance with a small application. They are non-toxic, non-polluting, and harmless to humans and livestock. Biostimulants play a key role in sustainable growth enhancement by increasing plant nutrient absorption and metabolic rates. A variety of biostimulants derived from algae, fungi, and bacteria have been used in agricultural production to improve crop yield and quality.

[0003] Microalgae are an abundant class of photosynthetic organisms found in freshwater and throughout marine systems. They include cyanobacteria and some eukaryotic algae, such as green algae, chlorophytes, and diatoms. Microalgae produce a wide variety of bioactive natural compounds, such as proteins, lipids, carotenoids, vitamins, and polysaccharides, and are a rich source of therapeutic agents for human diseases.

[0004] Chlorella fermentation technology can be used for large-scale biodiesel production, but the supernatant collected after fermentation still needs further treatment to meet emission standards. In agriculture, microalgae are mainly used in the form of living cells or cell extracts, and few studies have explored the application activities of extracellular metabolites of Chlorella protothecoides. Summary of the Invention

[0005] (1) Technical issues to be solved

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides the use of extracellular metabolites of Chlorella in promoting the growth and stress resistance of higher plants. This not only allows the supernatant wastewater from large-scale Chlorella cultivation to be further treated to meet wastewater discharge standards, but also utilizes the algae supernatant to provide a fertilizer additive with a biostimulant effect. This fertilizer additive can reduce the amount of fertilizer applied, promote plant growth and improve plant stress resistance. It has the advantages of being non-toxic, non-polluting, will not cause soil salinization, and is harmless to humans and livestock.

[0007] (2) Technical solution

[0008] In order to achieve the above objectives, the main technical solutions adopted by the present invention include:

[0009] In a first aspect, the present invention provides the use of extracellular metabolites of Chlorella as biostimulants in promoting growth and resisting stress in higher plants.

[0010] The application includes the application of the extracellular metabolites of Chlorella to improve the cold resistance of higher plants and promote the elongation of lateral roots.

[0011] Preferably, the higher plants include Arabidopsis thaliana and Nicotiana benthamiana.

[0012] Preferably, the application includes promoting the growth of vegetables, wherein the vegetables are rapeseed, tomato, cucumber, Chinese cabbage or lettuce. The extracellular metabolites of Chlorella can be used to increase the yield of rapeseed and increase the vitamin C and protein content in the vegetables.

[0013] Preferably, the Chlorella is Auxenochlorella protothecoides.

[0014] In a second aspect, the present invention provides a method for resource processing of the supernatant after harvesting the cultured Chlorella, comprising: sterilizing the supernatant with high-pressure steam and adding it to water and fertilizer as a plant stimulant, or freeze-drying the sterilized supernatant to make a powdered plant stimulant and adding it to solid fertilizer.

[0015] Preferably, the Chlorella is Chlorella protothecoides, the heterotrophic culture medium is a high nitrogen culture medium, and the culture mode is heterotrophic fermentation culture.

[0016] In a third aspect, the present invention provides a method for preparing a natural plant stimulant, wherein the plant stimulant is used to promote the growth of higher plants, wherein the promoting effects include enhancing the cold tolerance and stress resistance of higher plants, promoting lateral root growth, and accelerating the increase in stem diameter and stem height of seedlings. The preparation method comprises: performing cell separation on a culture solution of Auxenochlorella protothecoides, collecting the supernatant, and sterilizing the solution.

[0017] (3) Beneficial effects

[0018] The present invention uses three different culture strategies to prepare extracellular metabolites of Auxenochlorella protothecoides (EAp) and studies its effects on plant growth. EAp promotes lateral root elongation and cold tolerance in Arabidopsis thaliana and Nicotiana benthamiana. EAP produced by high-cell density fermentation, after being diluted 500-1000 times, has been shown to promote the growth of leafy vegetables such as rapeseed, tomatoes, cucumbers, cabbage, and lettuce. Experiments have found that EAP-treated rapeseed yields increased, and the vitamin C and protein content of the vegetables increased. This suggests that EAP can not only enhance vegetable cold tolerance (stress resistance), but also increase leaf vegetable yield and nutritional value, providing a new technological research and development direction for the cultivation of high-quality organic vegetables. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effect of the extracellular metabolite EAP of Chlorella protothecoides on the growth of Arabidopsis thaliana and Nicotiana benthamiana.

[0020] Figure 2 This is the effect of the extracellular metabolite Eap of Chlorella protothecoides on the growth of rapeseed.

[0021] Figure 3 Effects of concentrated EAP obtained under different cultivation strategies on plant growth.

[0022] Figure 4 The growth-promoting effects of EAP-1 and EAP-3 prepared in different cultivation modes (shake flask culture and fed-batch fermentation culture) on Brassica rapa (chicken feather vegetable) and Lactuca sativa (lettuce). DETAILED DESCRIPTION

[0023] The present invention cultivates Chlorella protothecoides and obtains its extracellular metabolite EAP from the algal liquid harvested from the algae. This metabolite is then used to promote plant growth and / or improve plant stress resistance. Experiments have confirmed that EAP indeed promotes lateral root elongation and improves the cold tolerance of Arabidopsis thaliana and Nicotiana benthamiana. EAP produced by high-cell density fermentation can still promote the growth of broccoli and lettuce after being diluted 500 to 1000 times. Therefore, the present invention provides a natural plant stimulant and a method for producing a plant stimulant by culturing microalgae using heterotrophic fermentation technology, which has the advantages of being environmentally friendly and economical. Compared with autotrophic cultivation, heterotrophic cultivation of Chlorella protothecoides has a higher cell density, a higher biomass, and easier-to-control culture conditions. The supernatant after harvesting also contains a higher content of the extracellular metabolite EAP.

[0024] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation methods in conjunction with the accompanying drawings.

[0025] Example 1

[0026] The microalgae strain used in this example is Auxenochlorella protothecoides 0710, and the preparation method of its extracellular metabolites is as follows:

[0027] 1. Preparation of heterotrophic culture medium HC and HN

[0028] The composition of the heterotrophic low nitrogen culture medium (HC) is: KH2PO4 0.7 g / L, K2HPO4 0.3 g / L, MgSO4 0.3 g / L, FeSO4.7H2O 3 mg / L, vitamin B1 10 μg / L, glucose 30 g / L, yeast extract 1 g / L, glycine 4 g / L, A5 trace element mother solution 1 ml / L, the formula of A5 trace element mother solution is H3BO3 286 mg / L, MnSO4·7H2O 250 mg / L, ZnSO4·7H2O 22.2 mg / L, CuSO4·5H2O 7.9 mg / L, Na2MoO4·2H2O 2.1 mg / L. After high temperature and high pressure sterilization, the heterotrophic Chlorella protothecoides culture medium can be obtained.

[0029] Heterotrophic high-nitrogen culture medium (HN) contained 5 g / L glycine and 2 g / L yeast extract, and other components were the same as those of HC medium.

[0030] 2. Prepare seed solution

[0031] A certain amount of cells of Auxenochlorella protothecoides 0710 were picked from the solid culture medium and inoculated into 20 mL of liquid HC and HN culture medium respectively. The cells were placed in a 28°C constant temperature shaker at 220 rpm in the dark for 4 days. 540 The protein content was 52.6%, which was much higher than that of Auxenochlorella protothecoides cultured in low nitrogen medium (HC, EAP-1). 540 The protein content is 10.3%.

[0032] 3. Preparation of EAP-1 and EAP-2 by shake flask culture

[0033] A certain volume of seed liquid was drawn and inoculated into 1 L of HC / HN liquid medium corresponding to the above seed liquid medium until the initial OD 540 The algae were collected when the residual glucose concentration was lower than 5 g / L. 540 The algae solution was centrifuged at 5000×g for 2 min to precipitate the algae cells, and the supernatant was sterilized by high-pressure steam at 108°C for 30 min to obtain EAP.

[0034] According to the different culture media, EAP from HC (low nitrogen medium) shake flask culture was recorded as EAP-1, and EAP from HN (high nitrogen medium) shake flask culture was recorded as EAP-2.

[0035] 3. Preparation of EAP-3 by fermentation in fermenter

[0036] The composition of the heterotrophic culture medium is: KH2PO4 4g / L, K2HPO4 1.6g / L, MgSO4.7H2O1.2g / L, FeSO4.7H2O0.01g / L, vitamin B1 15g / L, glucose 45g / L, yeast extract 1.6g / L, glycine 6g / L, A5 trace element mother solution 1ml / L, the formula of A5 trace element mother solution is H3BO3 286mg / L, MnSO4·7H2O 250mg / L, ZnSO4·7H2O 22.2mg / L, CuSO4·5H2O 7.9mg / L, Na2MoO4·2H2O 2.1mg / L. After high temperature and high pressure sterilization, the heterotrophic Chlorella protothecoides culture medium can be obtained.

[0037] A 5L fermenter (model: GBJS-5L-AUTOBIO, Zhenjiang Dongfang, China) was used for the fermentation of algae. The seed solution was inoculated, and the initial inoculation volume was OD 540 =6.8. The initial fermentation conditions were: temperature 28±0.5°C, pH 6.3, dissolved oxygen concentration (pO2) 100%, and stirring speed 300 rpm. Concentrated glucose and yeast extract were used for batch feeding, and the pH was adjusted to 6.5 using NaOH. During the fermentation process, the sucrose concentration was controlled at 8-25 g / L by manual feeding, and other parameters were automatically controlled. By adjusting the stirring speed and ventilation, pO2 was maintained above 20%. The cell density after 122 h of fermentation could reach a maximum of 34 (OD 540 ), the dry weight reached 60.1 g / L, more than three times that of shake flask culture. At this point, the algal solution was centrifuged at 5000 g for 2 minutes to pellet the algal cells. The supernatant was then autoclaved at 108°C for 30 minutes and designated EAP-3.

[0038] Example 2

[0039] The components of the extracellular metabolites EAP-1 and EAP-2 from Chlorella protothecoides obtained by the different methods described above were analyzed using GC-MS. In the following examples, one-way analysis of variance (ANOVA) was performed using GraphPad Prism 8.3.0 software to test differences between mean values. Post hoc t-tests were used to analyze significant differences between treatments; the significance level was set at P < 0.05.

[0040] Chromatographic analyses were performed using an Aglilent 8890-7010B GC–MS system (Agilent, USA) equipped with an HP-5ms capillary column (30 m × 250 μm ID, 0.25 μm film thickness; Agilent J&W Scientific, Folsom, CA). Samples (1 μl) were injected using an Agilent autoinjector with a 1:1 split ratio. Helium was used as the carrier gas at a flow rate of 1 mL / min. The injector temperature was set to 300°C. The GC oven temperature was heated to 60°C for 1 min, then increased to 80°C at 15°C / min, to 260°C at 10°C / min, to 280°C at 8°C / min, and then held at 325°C for 5 min. The ion source and source surface temperatures were set to 240°C and 280°C, respectively. Electron impact ionization (70 eV) was performed in full scan mode (m / z 50-800) at a rate of 20 scans / s. After a solvent delay of 4 min, the accelerating voltage was turned on. Ribitol was used as an internal standard to monitor the reproducibility of the batch and to correct for minor variations that occurred during sample preparation and analysis. Mass spectral data were acquired using gas chromatography-mass spectrometry real-time analysis software (Agilent, USA). The mass spectra of all detected compounds were compared with the Mass spectra were compared with those in the NIST library 2.4, an in-house mass spectral library maintained by the Plant Science Center. Nitrogen content was determined using a Kjeldahl nitrogen analyzer (Hanon, model K9840); phosphate content was determined using the quinoline phosphomolybdate gravimetric method. Gas chromatography-mass spectrometry was performed as described previously.

[0041] Analysis results:

[0042] To test the active ingredients in EAP, which play a key role in promoting growth and stress tolerance in higher plants, the contents of N, P, K, Fe, Mg, and free amino acids in EAP-1 and EAP-2 were measured. As shown in the table below, the organic matter content in EAP-1 and -2 was 0.12% and 1.18%, respectively, and the total nitrogen content was 0.42% and 0.31%, respectively. No boron or insoluble matter was detected in either EAP formulation. The organic matter and nitrogen contents in both EAP formulations were far lower than those in conventional fertilizers, and the contents of trace metals such as Fe, Zn, and B were also significantly lower than those in conventional fertilizers.

[0043]

[0044] EAP-2 was tested by GC-MS. Through library search, 14 compounds were identified in EAP-2 by negative ion mass spectrometry and 70 compounds were identified by positive ion mass spectrometry. Figure 1The main components of EAP-2 are organic acids or esters, phenols, saccharides, and other substances. Specifically, the compounds identified in EAP-2 include 50 organic acids or organic acid esters, 21 phenols, and 13 saccharides or 3 other compounds. Among them, the most abundant compounds are organic acids and organic acid esters, followed by phenols, glycosides, and saccharides. EAP-3 is the supernatant of a scaled-up culture in HN medium. Its composition is essentially the same as EAP-2, but its content is higher.

[0045] Example 3 Root promotion and stress resistance experiment

[0046] Root growth experiments were conducted on solid MS medium containing 30 g / L sucrose and 5% agar. A 1000-fold dilution of EAP1 was added to the MS medium. MS medium and MS medium containing a 1000-fold dilution of HC medium served as blank and negative controls, respectively.

[0047] Arabidopsis thaliana and Nicotiana benthamiana seeds were cultured on MS solid medium. After germination, the seeds were transferred to MS solid medium supplemented with varying volumes of EAP (1000-fold dilution of EAP-1), with six seedlings placed on each plate as biological replicates. Plates were positioned at a near-vertical angle to promote downward root growth. The culture temperature was 25°C, the light intensity was 2500 lux, and the light / dark cycle was 14 / 10 hours.

[0048] Experimental results:

[0049] See also Figure 1 As shown in the middle panel A, although EAP-1 has no significant effect on the length of the main root, it can significantly promote the length of lateral roots and the growth of fine fibrous roots on the lateral roots, and significantly improve the plant's ability to obtain water and nutrients, thereby increasing plant biomass and crop yield. H2O represents the blank control (MS medium without EAP), and HC represents the negative control (MS medium with 1000-fold diluted HC medium added). Therefore, EAP-1 does have the function of promoting the length of lateral roots and the growth of fine fibrous roots on lateral roots of Arabidopsis and Nicotiana benthamiana, while HC medium does not have the ability to promote the growth of lateral roots of Arabidopsis and Nicotiana benthamiana ( Figure 1 A).

[0050] See also Figure 1 B and Figure 1As shown in Figure C, in a cold stress experiment, each pot contained 1 kg of soil and an appropriate amount of water. Arabidopsis thaliana and Nicotiana benthamiana seeds were evenly sown in the soil. After 72 hours of imbibition, 7 mL of EAp was applied. The pots were placed in outdoor soil with a minimum nighttime temperature of 13°C. After 17 days, the control seedlings grew poorly, with leaves showing purple discoloration. Arabidopsis seedlings treated with EAP-1 maintained green leaves and grew significantly faster than the control seedlings treated with water. Seedlings treated with heterotrophic medium (HC) without algae culture died after 2 days of germination ( Figure 1 B and Figure 1 C) These results indicate that EAP-1 can enhance the resistance of higher plants to abiotic stresses.

[0051] Example 4: EAP treatment affects the growth and nutritional value of rapeseed

[0052] In the growth and nutritional value experiments, three rapeseed seedlings (e.g. Figure 2 A), 45mL or 90mL EAp-1 was applied 25 days after imbibition. H2O treatment was used as a blank control, and 7 replicates were set for each treatment. New leaves of similar size were marked on each plant before treatment. The leaf area of ​​the marked leaves was measured 25, 30, and 43 days before treatment and 5 and 18 days after treatment. Chlorophyll, protein, vitamin C content, leaf area, biomass, and root fresh weight were measured on the 43rd day. Leaf area was measured using a leaf area meter (LI-3100, LI-COR, Linkon, NE). Protein content was detected using the Coomassie Brilliant Blue method. Samples were digested with nitric acid, a color developer was added, and the absorbance at 400nm was measured. Vitamin C was detected using a C18 column LC-mass spectrometer. Final yield and root fresh weight were weighed using a balance.

[0053] 45mL and 90mL EAp-1 were applied to 25-day-old plants. After 5 days of treatment, the growth of the plants in the treatment group was significantly better than that in the control group. The leaf area of ​​the plants in the two different treatments increased by 26.8% and 29.3% respectively compared with the control group (the results are shown in Figure 2). Figure 2 At the same time, the yield of rapeseed (g / pot) was significantly increased compared with the control group (results as shown in Figure 2 C). Plants 18 days after treatment showed similar patterns.

[0054] like Figure 3 As shown, the fresh weight of the roots was measured and it was found that after treatment with 45 mL and 90 mL EAp-1, the fresh weight of the roots of rapeseed increased by 4.2% and 6.7%, respectively (the results are shown in Figure 2 D).

[0055] At the same time, the experiment also measured the vitamin C and protein content of rapeseed treated with EAP-1. The vitamin C (ascorbic acid) content of rapeseed treated with 45mL and 90mL EAp-1 increased by 3.4% and 6.9% respectively (the results are shown in Figure 2). Figure 2 E); after treatment with 90 mL EAp-1, the protein content increased by 21.4% (the results are shown in Figure 2 F).

[0056] These experimental results demonstrate that the application of EAP-1 can increase the yield of leafy vegetables, increase the fresh weight of roots, and enhance the vitamin C and protein content of vegetables. EAP-1 can enhance the nutritional value of leaves and can be used for the production of high-quality vegetables.

[0057] Example 5 Experiment on promoting vegetable growth

[0058] In this embodiment, soil (K413, Klasmann, Germany, without fertilizer) was used as the soil for plant cultivation in the following experiments.

[0059] Five tomato and cucumber seedlings germinated between two wet papers were transplanted into soil. EAP-2 was diluted 10-fold, 100-fold, and 500-fold. After the leaves emerged from the soil, they were treated with a series of diluted EAP-2. 50 days after transplanting, the height and diameter of the stems were measured using a ruler and a vernier caliper, respectively. Figure 3 ), tested the effects of extracellular metabolites of Auxenochlorella protothecoides 0710 cultured in media with two different nitrogen levels on plant growth. Figure 3 As shown in the serial dilution experiment, it was found that the stem diameter of Lycopersicum esculentum (tomato) treated with EAP-1 (marked as NO.1 in the figure) was smaller than that of the control ( Figure 3 B), while the stem height of Cucumis sativus (cucumber) treated with EAP-1 (marked as NO.1 in the figure) was 31% higher than that of the control group ( Figure 3 A).

[0060] Compared with the control group, the stem diameter and stem height of L. esculentum (tomato) increased by 32% and 12% after 100-fold dilution of EAP-1, and by 35% and 10% after 500-fold dilution of EAP-1. Figure 3 B and Figure 3 C) EAp-2 also produced similar growth-promoting effects when diluted 100-fold and 500-fold.

[0061] These results suggest that the functional components of EAP accumulate at higher levels in high-nitrogen medium and thus can serve as a concentrated biostimulant.

[0062] To test the effects of extracellular metabolites (EAp-1 and EAP-3) of Auxenochlorella protothecoides cultured in two different culture modes (i.e., shake flask culture and continuous fed-batch fermentation culture; shake flask culture is not suitable for large-scale industrial production) on plant growth, Auxenochlorella protothecoides 0710 was cultured in shake flasks and continuous fed-batch fermentation. Continuous fed-batch fermentation has higher production efficiency for industrial production of Auxenochlorella protothecoides and can achieve higher cell densities (e.g., 100% saturated fat) than shake flask culture. Figure 4 A). Use 2kg of soil to cultivate 9 cauliflower and lettuce seedlings in each pot (see cauliflower for details). Figure 4 B, lettuce Figure 4 C), After leaves emerged from the soil, 1 mL of 100-fold, 500-fold, and 1000-fold diluted EAP-1 and EAP-3 was applied to each plant.

[0063] After EAP-3 was diluted 100 times, 500 times and 1000 times, the effects of different concentrations of EAP-3 dilutions on plant growth were tested. Figure 4 As shown, EAP-3 (marked as NO.3 in the figure) showed similar effects on Brassica rapa ( Figure 4 B) and Lactuca sativa (lettuce) Figure 4 These results indicate that the fermentation supernatant of Auxenochlorella protothecoides can be used as a concentrated biostimulant.

[0064] Finally, it should be noted that although the specific embodiments of the present invention utilize Auxenochlorella protothecoides 0710 as an example for the cultivation of higher plants and vegetables, cultivation experiments with other Chlorella species have revealed no significant differences in the composition of extracellular metabolites among these species, with only minor fluctuations in content. Therefore, it can be inferred that replacing the experimental algae species with other Chlorella species will also produce similar plant stimulant properties for higher plants (and vegetables).

[0065] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of extracellular metabolites of Chlorella vulgaris as plant stimulants for improving cold tolerance and promoting lateral root elongation in higher plants, wherein the higher plants are Arabidopsis thaliana and Nicotiana benthamiana.

2. The use according to claim 1, characterized in that The chlorella is Auxenochlorella protothecoides.

3. A method for recycling the supernatant after harvesting Chlorella vulgaris, characterized in that: The supernatant is sterilized by high-pressure steam and added to water and fertilizer as a plant stimulant, or the sterilized supernatant is freeze-dried to make a powdered plant stimulant and added to solid fertilizer. The plant stimulant is used to improve the cold resistance of higher plants and promote lateral root elongation. The higher plants are Arabidopsis thaliana and Nicotiana benthamiana.

4. The resource recovery method according to claim 3, characterized in that: The chlorella is Auxenochlorella protothecoides, and the culture method is a high nitrogen medium under heterotrophic fermentation conditions.

5. A method for preparing a plant stimulant, wherein the plant stimulant is used to enhance the cold tolerance of Arabidopsis thaliana and Nicotiana benthamiana, promote lateral root growth, and accelerate the increase in stem diameter and stem height of tomato and cucumber seedlings; characterized in that: The preparation method comprises the following steps: performing cell separation on the culture solution of Auxenochlorella protothecoides, collecting the supernatant, and sterilizing the solution.

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