Application of Isaria endranulata 2018BY-1 in production of indoleacetic acid and promotion of plant growth

Abstract

The invention belongs to the technical field of microorganism application, and particularly relates to application of Isaria endula 2018BY-1 in production of indoleacetic acid and promotion of plant growth, the amount of indoleacetic acid secreted in a liquid LB culture medium containing L-tryptophan is 226.25 + / -25.98 mu g / mL, and the Isaria endula 2018BY-1 can effectively increase the content of indoleacetic acid in a plant environment to promote plant growth. Specifically, main root cells are induced to divide, propagate and extend, and the height of plants is increased, so that the leaf area of the plants is increased, the crop quality is improved, and the yield is increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial application, and specifically relates to the application of Cynospora annua 2018BY-1 in producing indoleacetic acid and promoting plant growth. Background Art

[0002] Tartary buckwheat (Fagopyrum tataricum) is a coarse grain crop that serves both medicinal and edible uses. Its seeds are highly sought after for their high content of protein, flavonoids, and trace elements. During their growth, buckwheat plants are often subject to various stresses, including fungal contamination, low free phosphorus levels in the soil, unsuitable pH, and drought. These stresses can directly lead to reduced germination rates and stunted plants. To mitigate the effects of these stresses, key measures include applying exogenous IAA and selecting or engineering crop varieties with the ability to stably synthesize IAA. However, artificial application of auxins can lead to soil and water pollution, ecosystem imbalance, and increased crop dependency. Furthermore, crop varieties with the ability to produce IAA are difficult to identify. Therefore, developing green, cost-effective, and efficient methods to increase crop IAA production is crucial for addressing crop IAA deficiencies, non-point source soil pollution, and maintaining ecological balance.

[0003] The IAA-producing fungi species currently found include Aspergillus awamori, Aspergillus flavus and Aspergillus niger, Fusarium asiaticum and Penicillium tumestinogenum, etc. For example, the patent publication number CN118440837A discloses a high-yield IAA-producing and stress-resistant growth-promoting bacterium Bacillus mirabilis YPR-35 and its application, but does not specifically disclose IAA production capacity. The patent publication number CN117481143A discloses the application of Paenibacillus polymyxa YF in the synthesis of indoleacetic acid IAA, but the IAA secretion of Paenibacillus polymyxa YF is not ideal, only 13.83 mg / L. Patent publication number CN118028122A discloses the use of Alternaria alstroemeriae J2 in producing IAA, but the IAA secretion of Alternaria alstroemeriae J2 is still unsatisfactory, at only 54.63 mg / L. Therefore, it is crucial to find a high-yield IAA strain, as it has become an effective means of using microorganisms to solve the growth and development problems of tartary buckwheat.

[0004] Isaria cateniannulata 2018BY-1 belongs to the subphylum Deuteromycotina, class Hyphomycetes, orders Hyphomycetales, and genus Cordyceps. It is a strain for which the applicant has applied for protection (publication number: CN118256356A, invention name: A phosphate-solubilizing Isaria cateniannulata and its application). This strain and its phosphate-solubilizing ability were first discovered in previous studies, but there are still not many reports on this strain. Therefore, in further research on its growth-promoting mechanism, it was unexpectedly discovered that it can secrete indoleacetic acid IAA. Since IAA is essential for plant growth, it is urgent to explore whether the reason for promoting plant growth is related to the ability of microorganisms to produce IAA. This provides us with a new way to develop efficient and green microbial fertilizers. Summary of the Invention

[0005] In response to the deficiencies in the prior art, the present invention proposes an application of Cynospora annua 2018BY-1 in promoting plant growth.

[0006] This is achieved specifically through the following technical solutions:

[0007] The first object of the present invention is to use Isaria cateniannulata 2018BY-1 in the production of indoleacetic acid, wherein the Isaria cateniannulata 2018BY-1 was deposited in the China Center for Type Culture Collection (CCTCC) on October 22, 2018, with a deposit number of CCTCC NO: M 2018698.

[0008] The production method comprises the following steps: inoculating the spores of the ringworm into a liquid LB medium containing L-tryptophan and culturing the medium for 5 days.

[0009] The concentration of the L-tryptophan is 200 mg / L.

[0010] The culture conditions are as follows: a rotation speed of 150 r / min, a temperature of 25° C., a humidity of 70%, a light intensity of 2000 lx, and a photoperiod of 12:12.

[0011] A second object of the present invention is to use Isaria cateniannulata 2018BY-1 in promoting plant growth. The Isaria cateniannulata 2018BY-1 was deposited in the China Center for Type Culture Collection (CCTCC) on October 22, 2018, with a deposit number of CCTCC NO: M 2018698. The deposit address is: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province (opposite the First Affiliated Primary School of Wuhan University). The function of promoting plant growth is achieved by producing indoleacetic acid.

[0012] The plant is buckwheat.

[0013] Furthermore, the application method for promoting plant growth is as follows: first, the spores of ... 7 The spore suspension is then added to the plant using any of the following methods: root irrigation, seed soaking, or spraying.

[0014] Furthermore, the application method for promoting plant growth is: first inoculate the spores of the spores into the liquid LB medium containing L-tryptophan and culture for 4 to 6 days to prepare a concentration of 2×10 7 / mL spore suspension, and then irrigate the roots of the plants during the seedling stage, irrigate twice a day in the morning and evening, and irrigate continuously for three days.

[0015] Beneficial effects:

[0016] The Cyclospora annua 2018BY-1 provided by the present invention has the ability to synthesize IAA, and the amount of indoleacetic acid secreted in a liquid LB culture medium containing L-tryptophan is 226.25±25.98 μg / mL. It can effectively increase the indoleacetic acid content in the plant environment to promote plant growth, specifically by inducing taproot cell division, reproduction and elongation, and increasing plant height, which is beneficial to increasing the leaf area of ​​the plant, thereby improving crop quality and increasing yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the IAA standard curve;

[0018] Figure 2 The color reaction of IAA produced by Cyclospora annuli; 1 represents Cyclospora annuli 2018BY-1, 5 represents Cyclospora annuli 08XS-1, and 8 represents Cyclospora annuli 19GZAI-1;

[0019] Figure 3 The effect of chain rod bundle on chlorophyll a of Tartary Buckwheat seedlings;

[0020] Figure 4 The effect of chain rod bundle on chlorophyll b in Tartary Buckwheat seedlings;

[0021] Figure 5 The effect of chain rod bundle on total chlorophyll of Tartary Buckwheat seedlings;

[0022] Figure 6 To study the effect of IAA-producing Cyclospora annularis on Tartary Buckwheat seedlings by root irrigation method;

[0023] Figure 7 The effect of IAA-producing Cyclospora annularis on Tartary Buckwheat seedlings in the seed soaking method;

[0024] Figure 8 This study is about the effects of IAA-producing Cyclospora annuli on Tartary Buckwheat seedlings by spraying. DETAILED DESCRIPTION

[0025] The specific embodiments of the present invention are further described in detail below, but the present invention is not limited to these embodiments. Any improvement or replacement based on the basic spirit of the present embodiment still falls within the scope of protection required by the claims of the present invention.

[0026] Example 1 Application of Cyclosporium annuum 2018BY-1 in the production of indoleacetic acid

[0027] 1 Materials and Methods

[0028] 1.1 Liquid culture medium preparation and strain culture

[0029] Liquid culture medium preparation: 5.0 g yeast powder, 10.0 g peptone, 10 g NaCl, were divided into 150 mL conical flasks, 50 mL per bottle, sterilized at 121 ° C for 15 min, and after the culture medium cooled to below 25 ° C, L-tryptophan was added to 200 mg / L, and deionized water was used to make up to 1 L to obtain liquid LB culture medium containing L-tryptophan (pH 5.6.0-6.0). 1 mL of 1 × 10 10 The bacterial liquid of Cyclosporus annuus 2018BY-1 with a spore count / mL was placed in a conical flask and cultured continuously for 5 days at a rotation speed of 150 r / min, a temperature of 25°C, a humidity of 70%, a light intensity of 2000 lx, and a photoperiod of 12:12.

[0030] At the same time, the Isariacateniannulata 08XS-1 strain disclosed in Publication No. CN104928185A and the Isariacateniannulate 19GAZ1-1 strain disclosed in Publication No. CN111876334A were cultured respectively using the same method.

[0031] 1.2 Standard curve drawing

[0032] Prepare concentrations of 0, 5.0, 10.0, 15.0, 20.0, and 25.0 μg / mL with ultrapure water. Pipette 2.0 mL of standard samples of different concentrations into 2.0 mL of Salkowskis reagent, mix thoroughly, and place in a light-shielded environment for 30 minutes before measuring OD. 530 The standard curve is drawn with the horizontal axis representing the IAA concentration level and the vertical axis representing the absorbance value. Figure 1 The IAA standard curve was y = 0.0032x, and the linear correlation coefficient R 2 =0.9996.

[0033] 1.3 Determination of IAA content in fermentation broth

[0034] 1.1 The fermentation broth was centrifuged at 5000 rpm for 10 min. 2.0 mL of the supernatant was aspirated and mixed with 2.0 mL of Salksowski colorimetric reagent (10 mL of 0.5 mol / L FeCl3 and 500 mL of 35% perchloric acid). The mixture was incubated in a dark water bath at 40°C for 30 min. The absorbance was measured at a wavelength of 530 nm using a UV spectrophotometer. Three replicates were set for each treatment. The data were substituted into the standard curve regression equation. The final IAA concentration was calculated as follows:

[0035] C=C1V1 / V2

[0036] Where: C is the sample indoleacetic acid concentration (μg / mL), C1 is the IAA concentration (μg / mL) obtained from the standard curve, V1 is the sample extract volume (mL), and V2 is the reaction volume (mL). A control group was prepared using liquid culture medium without inoculation.

[0037] 1.4 Data processing and analysis

[0038] Excel 2022 was used for basic data statistical analysis and the creation of bar and line charts. SPSS 24.0 software was used for statistical analysis. The independent sample T-test was used at the 0.05 level to analyze the significance of differences between two groups of data, and the Tukey method was used to analyze the significance of differences between multiple groups of data at the 0.05 level. Adobe Illustrator 2023 was used for image processing. Values ​​in the charts represent mean ± standard deviation. Lowercase letters represent the mean at the 0.05 level. The same letter indicates no significant difference between different methods, while different letters indicate significant difference between different methods. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Significant differences were observed between the experimental and control groups.

[0039] 2 Results and Analysis

[0040] 2.1 Screening and capacity analysis of IAA-producing Cyclospora spp. strains

[0041] In this experiment, the color of the 2018BY-1 strain of Corydalis annuli was significantly different from that of the CK group, Corydalis annuli 08XS-1, and Corydalis annuli 19GAZ1-1 using the color reaction of IAA with Salksowski's reagent. This strain was rescreened and its IAA production was quantitatively measured. The test results are shown in Table 1. As can be seen from Table 1, this strain has a high IAA production capacity, with an IAA yield of 226.25±25.98μg / mL.

[0042] Table 1 IAA content produced by Cyclosporium annuum

[0043]

[0044] Example 2 Application of Cyclospora annuli 2018BY-1 in Promoting Plant Growth

[0045] 1 Materials and Methods

[0046] 1.1 Liquid culture medium preparation and strain culture: same as Example 1.

[0047] 1.2 Field testing of the effects of Cyclospora annuli strains on buckwheat growth

[0048] 1.2.1 Inoculation of spores into the roots of buckwheat

[0049] Buckwheat planting: A plot experiment was conducted in a field with consistent water and fertilizer conditions (Buckwheat Industry Technology Center Experimental Base of Guizhou Normal University, Anshun City, Guizhou Province, located at 106°7′7″E, 26°5′9″N, and 1397m above sea level) from April to May 2024. Rice buckwheat No. 13 was selected as the experimental material. One plot was set up for each treatment, with 3 rows in each plot and 80 plants per row. Each treatment was replicated 3 times for a total of 3 m 2 The experimental group and the control group were separated by 1m, and the field management was exactly the same.

[0050] Experimental setup: Before planting the tartary buckwheat seeds treated by soaking method, 100 mL of spore suspension of strain No. 1 (2×10 7 spores / mL) for 4 hours before planting. For the buckwheat treated by root irrigation and spraying, at the seedling stage (15 days after planting), 100 mL (2×10 7 spores / mL), and the roots were irrigated and the front and back of the leaves were sprayed respectively. The control group CK was treated with liquid culture medium solution diluted at the same multiples for three consecutive days, with root irrigation once in the morning before sunrise and after sunset in the afternoon, for a total of 6 times.

[0051] 1.2.2 Determination of chlorophyll content

[0052] Determination of chlorophyll in buckwheat by alcohol extraction colorimetry: Take fresh buckwheat plant leaves, clean the dirt on the surface of the tissue, cut into pieces (remove the veins), and weigh 0.1g of the sample into a 25mL test tube. Then add 15mL of 95% ethanol solution to completely immerse the sample in the ethanol solution and soak it in the dark for 24h. Filter out the leaf fragments in the liquid to obtain the chlorophyll pigment extract. Pour the extract into a colorimetric dish, adjust the color to zero with 95% alcohol as a control, and measure the absorbance at wavelengths of 665nm and 649nm respectively. The method is based on Yang (Yang GM, Liu C, Gu LD, et al. Studies on the Phosphorus-Solubilizing Ability of Isaria cateniannulata and Its Influence on the Growth of Fagopyrum tataricum Plants [J]. Plants (Basel, Switzerland), 2024, 13 (12): 1694-1694) et al. The concentrations of chlorophyll a (Ca) and chlorophyll b (Cb) (where A is absorbance) and the content of each pigment per unit fresh weight of the tissue (in mg / g) were calculated using the following formula:

[0053] Ca=13.95×A665-6.88×A649

[0054] Cb=24.96×A649-7.32×A665

[0055] Chloroplast pigment content = (pigment concentration × extract volume) / sample fresh weight

[0056] 1.2.3 Determination of growth indicators of buckwheat

[0057] Determination of growth indicators of buckwheat: Buckwheat of the control group and experimental group were collected at the seedling stage, flowering stage, filling stage and maturity stage, with 10 plants in each row and 30 plants in each treatment. They were brought back to the laboratory and the plant height (cm) of buckwheat was measured from the first stem node above the root of the intact buckwheat plant using a soft ruler. The stem diameter (mm) at the fourth stem node from the root, the main root length (mm), the number of branches (branches), the number of stem nodes (nodes), the leaf area (cm) of the buckwheat plant were measured using a vernier caliper. 2 ), single plant weight (g), yield (kg / m 2 ) and other growth indicators.

[0058] 1.3 Data processing and analysis: Same as Example 1.

[0059] 2 Results and Analysis

[0060] 2.1 Effects of Cyclospora annuli on chlorophyll content in buckwheat seedlings

[0061] The chlorophyll a, chlorophyll b and total chlorophyll contents in the leaves of buckwheat seedlings inoculated with spores of spores of tartary buckwheat seedlings by root irrigation, seed soaking and spraying were significantly higher than those in the control group (P<0.001). The chlorophyll a content was 4.96±0.65 mg / g, 4.11±0.33 mg / g and 4.21±0.47 mg / g, respectively, which were 1.62, 1.26 and 1.35 times that of the control group. The total chlorophyll content was 711.34±1.34mg / g, 9.84±0.56mg / g and 9.21±0.99mg / g, which were 1.61, 1.42 and 1.31 times that of the control group, respectively. Figure 3-5 This is because Cyclospora annuli produces IAA, which, as an endogenous auxin in plants, can directly promote the elongation and division of leaf cells. By stimulating leaf expansion and increasing the photosynthetic area, the total amount of chlorophyll is indirectly increased (Chang Haixia, Li Jing, Li Mingyuan, et al. Screening of salt-tolerant indoleacetic acid (IAA)-producing multifunctional strains and their growth-promoting effects on salt-stressed wheat [J]. Journal of Jiangsu Agricultural Sciences, 2025, 41(01): 9-20.).

[0062] Depend on Figure 3-5 As shown, chlorophyll content in buckwheat plants significantly increased when inoculated with Isaria cateniannulata using root irrigation, seed soaking, and spraying (P < 0.05). However, the results varied between treatment methods. Soluble phosphorus content in buckwheat plants treated with root irrigation was significantly higher than that treated with seed soaking and spraying (P < 0.05), while no significant difference was found between seed soaking and spraying (P > 0.05). This is because spores of Isaria cateniannulata inoculated by root irrigation and spraying have a high survival rate and a much higher colonization rate in roots and leaves than in seeds. Furthermore, after colonizing the seeds, spores take longer to migrate to other parts of the plant, resulting in a reduced effectiveness (Xu Shaohuan. Effects of Isaria cateniannulata colonization on tobacco growth and rhizospheric microbial diversity [J]. Guizhou University, 2021.). Consequently, chlorophyll a, chlorophyll b, and total chlorophyll content in buckwheat leaves treated with root irrigation were significantly higher than those treated with seed soaking and spraying (P < 0.05). In summary, the effects of the three methods of inoculation with Cyclosporus phosphate-solubilizing on the chlorophyll content of buckwheat plants showed that root irrigation method > seed soaking method = seed soaking method.

[0063] 2.3 Effects of Cyclospora annuli on the growth of tartary buckwheat seedlings

[0064] Depend on Figure 6-8It can be seen that after inoculation with Isaria cateinannulata, the plant height of buckwheat seedlings was significantly higher than that of the control group (P<0.001) (Table 2). The experimental groups treated with root irrigation, seed soaking and spraying were 1.51, 1.72 and 1.68 times that of the control group, respectively. This is because Isaria cateinannulata can promote the growth of buckwheat (Peng Xue. Effect of Isaria cateinannulata colonization dynamics on enzyme activity and metabolites during buckwheat seed germination [D]. Guizhou Normal University, 2023.; Zhang, Xiaona, et al. "The colonization and effect of Isaria cateinannulata on buckwheat sprouts." Plants 12.1(2022):145.). The main root length of the experimental group was significantly higher than that of the control group (P<0.001) (Table 2). The experimental groups treated with root irrigation, seed soaking, and spraying were 1.23, 1.57, and 1.41 times that of the control group, respectively. This is related to the auxin secreted by the strain, which can cause the main root cells to elongate (Fang Fengguang, Sun Zhuo, He Feifei, et al. The growth-promoting effect of five rhizosphere growth-promoting bacteria producing auxin on Phaseolus vulgaris [J]. Journal of Science of Normal University, 2025, 45(03): 58-65), and vice versa. The level of auxin in the roots is a major limiting factor in plant growth, and factors such as root morphology, physiology, biochemistry, and symbiotic characteristics are related to the auxin content secreted by the plant (Li Ling. Handbook of Application of Plant Growth Regulators. Chemical Industry Press, 2013.). This also indirectly shows that the production of auxin by Cyclospora spp. induces the division, reproduction, and elongation of main root cells, thereby increasing the height of the plant. The growth and development of plant height and main root length are conducive to increasing the leaf area of ​​the plant, so that the leaf area of ​​the experimental groups treated with root irrigation, seed soaking and spraying methods increased by 1.89, 1.89 and 1.52 times that of the control group respectively.

[0065] Inoculation with Cyclosporus phosphate-solubilizing Tartary Buckwheat (Tartary Buckwheat) using different inoculation methods promoted growth. Plant height, taproot length, and leaf area differed significantly between the different methods (P < 0.05) (Table 2). The root irrigation method resulted in plant height that was 1.07 and 1.20 times greater than that of the seed soaking and spraying methods, respectively. The taproot length was 0.67 times greater, and the leaf area was 1.21 and 1.38 times greater than that of the seed soaking and spraying methods, respectively. This is because the colonization effect of Isaria cateniannulata inoculated by root irrigation is higher than that of seed soaking and spraying (Xu Shaohuan. Effects of Isaria cateniannulata colonization on tobacco growth and rhizospheric microbial diversity [J]. Guizhou University, 2021.), and excessive IAA produced by Isaria cateniannulata will affect the elongation of the main root (Fang Fengguang, Sun Zhuo, He Feifei, et al. The growth-promoting effect of five rhizospheric growth-promoting bacteria producing elongation on Phaseolus vulgaris [J]. Journal of Science of Normal University, 2025, 45(03): 58-65). In summary, the effects of root irrigation, seed soaking and spraying on the chlorophyll of buckwheat seedlings are different, that is, root irrigation > seed soaking = spraying.

[0066] Table 2 Effects of IAA-producing Cyclospora annularis on the growth of Tartary Buckwheat seedlings

[0067]

Claims

1. Application of Isaria cateniannulata 2018BY-1 in the production of indoleacetic acid, characterized in that: The described Cynospora annua 2018BY-1 was deposited in the China Center for Type Culture Collection (CCTCC) on October 22, 2018, with a deposit number of CCTCC NO: M 2018698.

2. The use according to claim 1, characterized in that The production method is to inoculate the spores of the spores 2018BY-1 into a liquid LB medium containing L-tryptophan and culture for 5 days.

3. The use according to claim 2, characterized in that The concentration of the L-tryptophan is 200 mg / L.

4. The use according to claim 2, characterized in that The culture conditions are as follows: a rotation speed of 150 r / min, a temperature of 25° C., a humidity of 70%, a light intensity of 2000 lx, and a photoperiod of 12:

12.

5. Use of Isaria cateniannulata 2018BY-1 in promoting plant growth, characterized in that: The described Cynospora annua 2018BY-1 was deposited in the China Center for Type Culture Collection (CCTCC) on October 22, 2018, with a deposit number of CCTCC NO: M 2018698; the function of promoting plant growth is achieved by producing indoleacetic acid.

6. The use according to claim 5, characterized in that The plant is buckwheat.

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