CN115044501A - Endophytic rare actinomycetes for promoting plant growth and application thereof - Google Patents
Endophytic rare actinomycetes for promoting plant growth and application thereof Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/06—Coating or dressing seed
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
Abstract
The invention discloses an endophytic rare actinomycete for promoting plant growth and application thereof, wherein the endophytic rare actinomycete is Amycolatopsis sp.MtRT-6 and has a strain preservation number of CGMCC No. 23411. The rare actinomycetes can be used in spore suspension form for promoting growth of plants of different families, such as leguminous plants and gramineous plants. The endophytic rare actinomycetes can enter different families of plants (such as leguminous plants and gramineae plants) for colonization, has unique ecological niches as endophytic bacteria, has more direct effect on the plants than soil and rhizosphere microorganisms, can not cause damage to the growth and development of the plants, can remarkably promote the growth of the plants in the whole growth cycle, can fully play the role of promoting the growth of the plants particularly in the initial growth stage of the plants, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of crop planting, and relates to an endophytic rare actinomycete for promoting plant growth and application thereof.
Background
Alfalfa is a perennial bean herbaceous plant, is a very important member of crops, and has far better adaptability, economic value and ecological value than other pastures. At present, the demand for alfalfa is vigorous, but the quality of alfalfa products is low, so that the method for seeking to improve the yield of high-quality alfalfa is very important. Rice is one of the most important grain crops, and a new technical approach for increasing yield in an environment-friendly manner is found to be urgent in order to meet the increasing demand of the world on grain supply.
Endophytes are a class of microorganisms that are closely related to plants. Endophytes are generally considered to be groups of microorganisms that live within the tissues of the host plant at some or all of their life history and do not cause significant disease symptoms to the plant. In nature, endophytes are widely present in almost all known plants, and a variety of microorganisms may co-exist in one plant at the same time, and may co-evolve with the plant to form a more coordinated reciprocal symbiotic relationship. For example, Heena et al report that endophytes isolated from Gnetum gnemon L. promote tomato growth by seedling root inoculation. Compared with fungi and bacteria, actinomycetes can produce abundant secondary metabolites, and are important producers of bioactive secondary metabolites such as antibiotics, enzymes, enzyme inhibitors and antioxidants. Based on this, some reports have been made on the use of endophytic actinomycetes for promoting plant growth, but most of them focus on the genus Streptomyces. For example, Juliana et al report that endophytic actinomycetes Streptomyces sp.CLV45 can promote the growth of soybean seedlings by a method of soaking soybean seeds in spore liquid; in addition, Thladete et al report that an endophytic actinomycete Streptomyces sp.JJY4T is inoculated with spores of JJY4T at the bottom of a pot, so that the growth of cacao seedlings can be promoted; streptomyces sp.MR14 reported by Talwinder et al can help tomato to inhibit blight caused by Fusarium moniliforme and promote tomato plant growth; subramaniam et al reported five endophytic actinomycetes, Streptomyces spp. CAI-24, CAI-121, CAI-127, KAI-32 and KAI-90, which all increased sorghum and rice yields; le et al reported that the dry weight of the aerial parts of alfalfa was increased by seed embedding the spore liquid of Streptomyces spp. LuP30 and LuP 47B.
Unlike the common streptomyces, amycolatopsis is a rare actinomycete. There are 82 species of amycolatopsis that have been published, most of which are isolated from soil and marine sediments. However, the application of amycolatopsis is mostly focused on the production of antibiotics and the degradation of chemicals. For example, patent No. CN201710609367.7 by Zhao Hai soldier et al, which confirmed the culture medium and culture method for producing rifamycin by fermentation using Amycolatopsis mediterranei; wanghong et al (CN201610231668.6) use the bacterial liquid of Amycolatopsis to degrade the quinestrol, and degrade 50mg/L of the quinestrol by 100% in 5 days; the invention patent (CN202111206808.1) of Navie et al shows a method for degrading phosphotriester (2-chloropropyl) by amycolatopsis and application thereof; the patent (CN202111070795.X) of Meng Yonghong et al relates to a construction method and application of gene-deficient amycolatopsis for high-yield vanillin, and provides application of deletion of ROK family transcription regulation genes ROK-TR1, ROK-TR2 or ROK-TR3 in improvement of vanillin production of amycolatopsis.
Amycolatopsis strains have been reported to have plant growth promoting ability, however, these Amycolatopsis strains were isolated from plant rhizosphere and belong to rhizosphere beneficial bacteria. To date, few specific reports have been made on amycolatopsis isolated from the interior of plants as a rare actinomycete endogenously produced by plants and used to promote the growth of plants of different families.
Therefore, the research on the utilization of the amycolatopsis to promote the growth of different families of plants enriches the beneficial microorganism resource library, provides scientific basis for promoting the wide application of the amycolatopsis in agriculture, and has important significance.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide an endophytic rare actinomycete capable of promoting the growth of different families and application thereof, wherein the endophytic rare actinomycete can enter different families to colonize, is used as an endophytic bacterium, has a unique ecological niche, has a more direct effect on plants than soil and rhizosphere microorganisms, cannot damage the growth and development of the plants, can obviously promote the growth of the plants in the whole growth period, particularly can fully play the role of promoting the growth of the plants in the early growth stage of the plants, and has a good application prospect.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
an endophytic rare actinomycete for promoting plant growth is Amycolatopsis sp.MtRT-6, and the strain preservation number is CGMCC No. 23411.
In the above mentioned rare actinomycetes, further improvement, said rare actinomycetes is obtained by separating and purifying the root of alfalfa.
As a general technical concept, the invention also provides an endophytic rare actinomycete spore suspension which is obtained by culturing the endophytic rare actinomycete.
The above-mentioned endophytic rare Actinomyces spIn a further improvement, the concentration of the suspension of spores of the rare-earth endophyte is 1 x 10 5 CFU/mL～1×10 8 CFU/mL。
As a general technical concept, the invention also provides the application of the endophytic rare actinomycete spore suspension in promoting the growth of leguminous plants and/or gramineous plants.
The application is further improved and comprises the following steps: planting seedlings of leguminous plants and/or gramineous plants in soil, and irrigating an endophytic rare actinomycete spore suspension into the soil where roots of the leguminous plants and/or gramineous plants are located, and cultivating the seedlings of the leguminous plants and/or gramineous plants.
In the application, the filling amount of the endophytic rare actinomycete spore suspension is 1 mL/strain; the concentration of the endophytic rare actinomycete spore suspension is 1 multiplied by 10 5 CFU/mL～1×10 7 CFU/mL; the leguminous plant is alfalfa; the gramineous plant is rice.
The application is further improved, and comprises the following steps: the seeds of leguminous plants and/or gramineae plants are embedded by utilizing the endophytic rare actinomycete spore suspension, and the leguminous plants and/or gramineae plants with the surface coated with the endophytic rare actinomycete are cultivated.
In the above application, it is further improved that, before the embedding treatment, the method further comprises mixing the endophytic rare actinomycete spore suspension with an embedding agent; the proportion of the leguminous plant and/or gramineous plant seeds, the embedding medium and the endophytic rare actinomycete spore suspension is 1g to 463 mu L to 51 mu L; the embedding agent is xanthan gum; the volume fraction of the xanthan gum is 0.3%; the concentration of the endophytic rare actinomycete spore suspension is 1 multiplied by 10 6 CFU/mL～1×10 8 CFU/mL。
In the above application, further improvement, the seeds of the leguminous plants are alfalfa seeds; the gramineous plant seed is a rice seed.
Compared with the prior art, the invention has the advantages that:
(1) aiming at the defect that the existing Amycolatopsis strain can not be used for simultaneously promoting the growth of plants of different families, the invention creatively provides an endophytic rare actinomycete capable of promoting the growth of plants of different families, wherein the endophytic rare actinomycete is Amycolatopsis sp.MtRT-6, which is preserved in the China general microbiological culture Collection center (CGMCC for short) and has the address of No. 3 Hopkin Xilu No.1 of the sunward area in Beijing, and the strain preservation number is CGMCC No.23411, which is a plant endophytic rare actinomycete obtained after the separation and purification of the root of alfalfa. Unlike other conventional amycolatopsis, the endogenous rare actinomycetes (hereinafter abbreviated as MtRT-6) of the present invention have nitrogen-fixing, phosphorus-dissolving and potassium-dissolving capabilities which can help improve the absorption and utilization of nitrogen, phosphorus and potassium elements necessary for growth and development of host plants, and MtRT-6 can also produce ACC deaminase which can help plants resist external stress by regulating ethylene in plants to promote plant growth, and at the same time, hydrolase such as protease and cellulase produced by MtRT-6 can help it enter plant tissues to colonize in plants, and MtRT-6 also produces chitinase which can degrade fungal cell walls, one of the properties of probiotics indirectly promoting host plant growth by antagonizing pathogenic bacteria, and MtRT-6 can produce auxin (IAA), Gibberellin (GA) and 6-benzyladenine (6-BA) which are associated with plant growth As can be seen, the MtRT-6 can produce various active substances, has the function of promoting the growth of different families of plants, and has important significance for simultaneously promoting the growth of different families of plants and improving the yield of different families of plants.
(2) The endophytic rare actinomyces Amycolatopsis sp.MtRT-6 grows well on most culture media, can grow well in the environment with the pH value of 6.0-12.0, can utilize various carbon sources such as glucose and arabinose, has strong growth capacity and is easy to culture. In addition, secondary metabolites such as relevant hormones for promoting plant growth can be secreted, and compared with a chemical fertilizer, the biological organic fertilizer is more stable, safe and environment-friendly.
(3) The invention uses the endophytic rare actinomycete spore suspension obtained by culturing the endophytic rare actinomycete to perform root irrigation or embedding treatment on leguminous plants (such as alfalfa seedlings) or gramineous plants (such as rice), the strain can enter the leguminous plants or gramineous plants to colonize, not only can not damage the growth and development of the plants, but also can remarkably promote the growth of the plants in the whole growth period, and particularly can fully play the role of promoting the growth of the plants in the early growth stage.
(4) When the endophytic rare actinomycete spore suspension is used for promoting the growth of leguminous plants and gramineous plants, the related application method is more convenient and simpler, and compared with other land management methods, the method saves time and labor, is not limited by regions and manpower, is suitable for wide popularization, and simultaneously, compared with chemical fertilizers, the method is more environment-friendly, safer for human beings and beneficial to the sustainable development of agriculture.
Drawings
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.
FIG. 1 is a phylogenetic tree of an endophytic rare actinomycete strain according to the 16S rRNA gene sequence in example 1 of the present invention.
FIG. 2 is a colony morphology of the strain of rare actinomycete endogenously in example 1 of the present invention on different types of media.
FIG. 3 is a graph showing the effect of the spore suspension of an endophytic rare actinomycete on the growth of alfalfa seedlings in example 2 of the present invention, where E-is the control group that was not inoculated with MtRT-6 and E + is the treatment group that was inoculated with MtRT-6.
FIG. 4 is a graph showing the effect of the spore suspension of an endophytic rare actinomycete on the development of roots of alfalfa seedlings in example 2 of the present invention, where E-is the control group that was not inoculated with MtRT-6 and E + is the treatment group that was inoculated with MtRT-6.
FIG. 5 is a graph showing the effect of the suspension of endospores of rare actinomycetes in example 3 of the present invention on the growth of rice seedlings, in which E-represents a control group not inoculated with MtRT-6 and E + represents a treatment group inoculated with MtRT-6.
FIG. 6 is a graph showing the effect of the suspension of spores of an endophytic rare actinomycete on the development of roots of rice seedlings in example 3 of the present invention, in which E-represents a control group in which MtRT-6 was not inoculated and E + represents a treatment group in which MtRT-6 was inoculated.
FIG. 7 is a graph showing the effect of the spore suspension of the endophytic rare actinomycetes of example 4 of the present invention on rice yield under field planting conditions, wherein E-is a control group which was not inoculated with MtRT-6, and E + is a treatment group which was inoculated with MtRT-6.
Detailed Description
The invention is further described below with reference to the drawings and the specific preferred embodiments, without thereby limiting the scope of protection of the invention.
In the following examples, unless otherwise specified, the raw materials and equipment used are commercially available, and the process used is a conventional process and the equipment used is conventional equipment.
Example 1
An endophytic rare actinomycete for promoting plant growth is Amycolatopsis sp.MtRT-6 with a preservation number of CGMCC No.23411 and a preservation date of 2021, 9 and 15 and is preserved in the China general microbiological culture Collection center. The 16S rRNA gene sequence of the endogenous rare actinomycetes is shown in a sequence table SEQ ID NO. 1.
The endogenic rare actinomycete Amycolatopsis sp. MtRT-6 (abbreviated as MtRT-6) in the present example is an actinomycete isolated from the roots of perennial alfalfa after surface sterilization. Perennial alfalfa was collected in 2019 in 10 months in Germany's pasture science and technology limited base (29 ° 7'18"N, 112 ° 4'21" E), sample variety WL525, autumn sleep grade 8.2, belonging to non-autumn sleep type alfalfa, produced in the United states.
The physiological identification and physiological and biochemical characteristics of the endophytic rare actinomycetes MtRT-6 in the embodiment are as follows:
(1) MtRT-6 isolation and screening procedure
Cleaning an acquired alfalfa sample, airing, cutting into small sections, carrying out surface disinfection, placing the sample on an isolation medium (yeast extract medium) after the sample is dried, culturing for 14 days at 30 ℃, transferring the separated actinomycetes onto a PDA solid medium for culturing and purifying to obtain the endophytic rare actinomycetes MtRT-6 for subsequent identification.
(2) MtRT-6 molecular biological identification
In the experiment, DNA of the MtRT-6 is extracted as a template, 16S rRNA is amplified by two pairs of bacteria universal primers 27f-765r and 704f-1402r respectively, the obtained products are sequenced and spliced to obtain 16S rRNA of the MtRT-6, the sequence length of the 16S rRNA is 1425bp, and the 16S rRNA gene sequence of the endogenous rare actinomycetes (MtRT-6) is shown as a sequence table SEQ ID NO. 1. This sequence was uploaded to the ezbiochloud website for homology alignment of model strains and used to map phylogenetic trees of MtRT-6 and related strains using MEGA7.0 software in a neighborhood approach (see FIG. 1) to find their homology to Amycolatopsis rhabdoformis SB026 T The homology was highest (98.52%). By further examining Bergey's Manual of bacteria and finding the literature, the physiological and biochemical characteristics of MtRT-6 were found to be related to the strain Amycolatopsis rhabdoformis SB026 T With a greater number of differences (see table 1). MtRT-6 is able to utilize more of a different carbon source than Amycolatopsis rhabdoformis SB026 T Then, for example, glucose and arabinose, etc. cannot be used. In addition, the growth temperature and salinity tolerance of MtRT-6 were higher than those of Amycolatopsis rhabdoformis SB026 T . Thus, combining the physiological and biochemical characteristics and 16S rRNA sequence homology analysis, the rare endophytic actinomycetes was named Amycolatopsis sp.
TABLE 1 Amycolatopsis sp. MtRT-6 strain and Amycolatopsis rhabdoformis SB026 in example 1 of the present invention T Comparison of physiological and biochemical characteristics and carbon source utilization conditions
Note: + indicates a positive result; ± represents a weak positive result; -indicating a negative result; ND means not determined.
(3) Physiological and biochemical identification of MtRT-6
MtRT-6 was inoculated on different media, respectively, as shown in FIG. 2, MtRT-6 grew well, with best vigor on ISP2 medium, abundant spores were produced, aerial hyphae appeared white, and intrastromal hyphae were colorless. Referring to Table 1, MtRT-6 was inoculated into ISP2 liquid medium and cultured at various temperatures (10-45 ℃), and it was found that MtRT-6 could not grow at all under the culture conditions of 10 ℃ and 45 ℃ and the suitable growth temperature was around 30 ℃; inoculating into ISP2 liquid culture medium with different NaCl concentrations (1-8%), and growing at NaCl concentration of 7% or below; inoculated into a basal medium containing 1% of different carbon sources, and it was found that, in addition to xylose, MtRt-6 can utilize various carbon sources such as glucose and arabinose. In conclusion, from all aspects of physiological characteristics of the MtRT-6, the adaptability of the MtRT-6 to growth environments is high.
MtRT-6 has nitrogen fixing, phosphorus dissolving and potassium dissolving capabilities, and the capabilities can help improve the absorption and utilization of nitrogen, phosphorus and potassium elements necessary for growth and development of host plants. MtRT-6 also produces ACC deaminase, which can help plants resist external stress and promote plant growth by regulating ethylene in plants. In addition, the hydrolase such as protease, cellulase and the like produced by MtRT-6 can help it enter plant tissues and colonize in plants. MtRt-6 also produces chitinases that degrade the fungal cell wall, one of the properties of probiotics in indirectly promoting host plant growth by antagonizing pathogenic bacteria. Further, the metabolites of MtRT-6 were examined and found to be capable of producing plant growth-related hormones such as auxin (IAA), Gibberellin (GA), and 6-benzyladenine (6-BA) (see Table 2). In conclusion, MtRT-6 can produce various active substances and has the potential of promoting the growth of different families of plants.
TABLE 2 growth promoting Properties and detection results of active substances produced by Amycolatopsis sp.MtRT-6 strain in example 1 of the present invention
| Enzyme and secondary metabolite detection | MtRt-6 |
| Nitrogen fixation capacity | + |
| Phosphorus dissolving capacity | + |
| Potassium decomposing ability | + |
| ACC deaminase | + |
| Growth hormone | + |
| Gibberellins | + |
| 6-benzyladenine | + |
| Protease enzyme | + |
| Cellulase enzymes | + |
| Chitinase | + |
Note: + indicates a positive result.
Whole genome sequencing analysis of MtRT-6 was performed to excavate 19 nitrogen metabolism-related genes from the MtRT-6 genome (see Table 3), and to determine that MtRT-6 could grow on nitrogen-free medium, presumably with nitrogen fixation capacity and with some advantage in nitrogen metabolism. Further analyzing genes related to growth promotion, and excavating 17 genes related to phosphorus dissolving. In addition, there are 14 genes associated with auxin synthesis in the MtRT-6 genome. These results all suggest that MtRT-6 has the potential to promote plant growth.
TABLE 3 genes associated with growth promotion in the genome of Amycolatopsis sp. MtRT-6 strain in example 1 of the present invention
Example 2:
an endogenic rare actinomycete spore suspension obtained by culturing the endogenic rare actinomycete (Amycolatopsis sp. MtRT-6) of example 1 at a concentration of 1X 10 6 CFU/mL, comprising the steps of:
(1) the strain of rare actinomyces endophytic (Amycolatopsis sp. MtRT-6) in example 1 was inoculated onto ISP2 solid medium and cultured for 7 days at 30 ℃ in an incubator.
(2) And (3) dipping a sterile cotton swab in sterile water, slightly dipping the spores on the surface of the mycelium in the step (1), and rinsing the spores into an EP (EP) tube filled with the sterile water.
(3) Counting the spore suspension obtained in step (2) by using a blood counting plate to adjust the concentration to 1 × 10 6 CFU/mL to obtain the endophytic rare actinomycete spore suspension.
An application of the endobiotic rare actinomycete spore suspension in the embodiment in promoting growth of leguminous plants, specifically, the method for promoting growth of medicago truncatula by using the endobiotic rare actinomycete spore suspension includes the following steps:
preparing sterilized nutrient soil and vermiculite (volume ratio is 1: 1), planting alfalfa seedling, and then filling the alfalfa seedling root with the concentration of 1 multiplied by 10 6 CFU/mL endophytic rare actinomycete spore suspension, using 1 mL/seedling (E +), culturing alfalfa seedlings, wherein the planting mode is greenhouse soil culture, and the greenhouse conditions are as follows: 16 hours light/25 ℃, 8 hours dark/20 ℃. The control group was made by pouring the same amount of water (E-) into the roots of alfalfa seedlings. 10 replicates per treatment group.
And (3) effect statistics: and (5) counting the dry weight of the alfalfa, the total chlorophyll content, the root activity, the root morphology index and the nitrogen content after culturing for 40 days.
And (3) test results: the E + alfalfa seedlings treated with the MtRT-6 spore suspension by root drenching had significantly better growth vigour than the E-control seedlings (see FIG. 3). Compared with the E-seedling, the dry weight, total chlorophyll content, root activity and nitrogen content of the E + seedling were significantly increased by 64.49%, 18.01%, 26.48% and 4.79%, respectively (see Table 4).
TABLE 4 Effect of Amycolatopsis sp. MtRT-6 spore suspension on alfalfa seedling growth
| Dry weight/mg | Total chlorophyll content/SPAD | Root activity/mg g -1 h -1 | Nitrogen content/mg g -1 | |
| E- | 19.71±1.512 | 30.12±1.028 | 168.33±7.36 | 5.01±0.015 |
| E+ | 32.43±2.418*** | 35.54±0.476*** | 212.90±8.80* | 5.25±0.001*** |
Indicates that the difference between E-and E + alfalfa was significant (ANOVA, # p <0.001, # p <0.05), based on Tukey-Kramer test.
The root development of E + alfalfa seedlings was significantly better than that of E-control seedlings (see FIG. 4). Further statistics and analysis showed that the total root length, root projected area, root surface area, root volume and number of root tips of the E + seedlings were significantly increased by 250.91%, 293.11%, 293.11%, 340.12% and 301.53%, respectively, compared to the E-seedlings (see table 5). Thus, the endophytic rare actinomycetes (Amycolatopsis sp. MtRT-6) of the present invention can promote the growth of alfalfa seedlings.
TABLE 5 Effect of Amycolatopsis sp. MtRT-6 spore suspension on root growth of alfalfa seedlings
| Total root length/cm | Root projected area/cm 2 | Root surface area/cm 2 | Root volume/cm 3 | Root and tip/number | |
| E- | 83.75±3.32 | 2.98±0.19 | 9.37±0.60 | 0.084±0.01 | 65.5±13.5 |
| E+ | 293.90±6.09*** | 11.72±0.047*** | 36.83±0.15*** | 0.37±0.00*** | 263±17*** |
Indicates that the difference between E-and E + alfalfa was significant (ANOVA,. p <0.001), based on Tukey-Kramer test.
Example 3:
an endogenic rare actinomycete spore suspension obtained by culturing the endogenic rare actinomycete (Amycolatopsis sp. MtRT-6) of example 1 at a concentration of 1X 10 7 CFU/mL, comprising the steps of:
(1) the strain of rare actinomyces endophytic (Amycolatopsis sp. MtRT-6) in example 1 was inoculated onto ISP2 solid medium and cultured for 7 days at 30 ℃ in an incubator.
(2) And (3) dipping a sterile cotton swab in sterile water, slightly dipping the spores on the surface of the mycelium in the step (1), and rinsing the spores into an EP (EP) tube filled with the sterile water.
(3) Counting the spore suspension obtained in step (2) by using a blood counting plate to adjust the concentration to 1 × 10 7 CFU/mL to obtain the endophytic rare actinomycete spore suspension.
The application of the endophytic rare actinomycetes spore suspension in the embodiment in promoting growth of gramineous plants, in particular to promoting growth of rice (indica rice 93-11) by using the endophytic rare actinomycetes spore suspension, comprises the following steps:
according to the proportion of 1 g: 463 mu L: 51 mu L of grass family plant seeds, embedding agent and endophytic rare actinomycetes spore suspension, mixing the endophytic rare actinomycetes spore suspension with xanthan gum with volume fraction of 0.3% (v/v), embedding (E +) the disinfected rice seeds, then carrying out germination accelerating treatment on the rice seeds coated with the endophytic rare actinomycetes spore suspension, and finally transferring the seeds after germination accelerating to water culture nutrient solution (0.3mM KH) 2 PO 4 ，0.35mM K 2 SO 4 ，1mM MgSO 4 ·7H 2 O，0.5mM Na 2 SiO 3 ·9H 2 O，1mM CaCl 2 ·2H 2 O，9μM MnCl 2 ·4H 2 O，20μMH 3 BO 3 ，0.77μM ZnSO 4 ·7H 2 O，0.32μM CuSO 4 ·5H 2 O，20μM NaFeEDTA，0.39μMNa 2 MoO 4 ·2H 2 O, pH 5.8) were cultured in an incubator, and the hydroponic medium was changed every 2 days. The specific culture conditions are as follows: fluorescent lamp illumination (300- -2 ·s -1 ) (ii) a Long day conditions (16 hours light/8 hours dark); the temperature is 30 ℃; the relative humidity is 60-70%. After 14 days of culture, the relevant physiological indicators were measured. As a control (E-), rice seeds/0.3% (v/v) xanthan gum/sterile water ratio was 1 g/463. mu.L/51. mu.L. Each treatment was repeated in 3 groups, each group comprising 48 rice seedlings.
And (3) effect statistics: and (4) counting the dry weight of the seedlings, the activity of the root system, the root morphology index and the nitrogen content of the roots after 14 days of water culture.
And (3) test results: under hydroponic conditions, the growth status of E + rice seedlings embedded in MtRT-6 spore suspension was significantly better than that of the E-control group (see FIG. 5). The dry weight, total chlorophyll content, root vigor and root nitrogen content of the E + seedlings were significantly increased by 17.73%, 12.05%, 14.41% and 8.62%, respectively, over those of the E-seedlings (see table 6).
TABLE 6 Effect of Amycolatopsis sp. MtRT-6 spore suspension on growth of Rice seedlings
| Dry weight/mg | Root activity/mg g -1 h -1 | Total chlorophyll content/mg g -1 FW | Nitrogen content of root system | |
| E- | 61.84±2.387 | 961.61±9.549 | 0.79±0.006 | 1.83±0.021 |
| E+ | 72.81±2.944** | 1100.21±28.228*** | 0.88±0.008** | 1.99±0.045* |
Indicates that the difference between E-and E + rice was significant (ANOVA, <0.001, <0.01, <0.05), based on Tukey-Kramer test.
Meanwhile, the root development of the E + rice seedlings is also remarkably promoted (see fig. 6), in fig. 6, A is a rice root scanning image, and B is a rice root scanning electron microscope image, wherein the total root length, the root projection area, the root surface area, the root volume and the number of root tips of the seedlings are respectively and remarkably improved by 39.21%, 43.84%, 43.85%, 45.03% and 71.31% compared with those of the E-seedlings (see table 7). Therefore, the endophytic rare actinomycetes (Amycolatopsis sp. MtRT-6) of the present invention can promote the growth of rice seedlings.
TABLE 7 Effect of Amycolatopsis sp. MtRT-6 spore suspension on root growth of Rice seedlings
| Total root length/cm | Root projected area/cm 2 | Root surface area/cm 2 | Root volume/cm 3 | Root and tip/number | |
| E- | 110.39±3.92 | 5.32±0.75 | 16.71±2.35 | 0.21±0.05 | 437.50±32.25 |
| E+ | 153.67±4.68*** | 7.65±0.18*** | 24.04±0.58*** | 0.30±0.01** | 749.50±70.62*** |
Indicates that the difference between E-and E + rice was significant (ANOVA, # p <0.001, # p <0.01), based on Tukey-Kramer test.
Example 4:
under the field environment, the MtRT-6 is used for embedding rice seeds to promote the growth of rice
The planting mode is as follows: the rice treated differently was sown in experimental fields of Longping Gaokouke (Changsha Guanshan research and development base) at 3 months 2021.
And (3) test crops: rice (indica 93-11).
Experimental treatment: a spore suspension of MtRT-6 was prepared according to the method of example 3, and the rice seed embedding treatment was performed according to the method of example 3, with the treatment group being E + and the blank group being E-. Each group repeats 3 cells. After the rice is mature, the corresponding agronomic index is determined.
And (3) effect statistics: after the rice is mature, the effective tillering number, the ear length, the single plant yield and the thousand seed weight of the rice are counted.
Under field conditions, after the rice in the E + treatment group in which the MtRT-6 spore suspension liquid is embedded with rice seeds is matured, the growth condition of the rice is obviously superior to that of the E-control group. As shown in fig. 7, the ears of E + rice were larger and the tillers were more numerous. Through specific data statistical analysis, the effective tiller number, ear length, single plant yield and thousand kernel weight of the E + rice are respectively and obviously improved by 47.70%, 7.04%, 61.85% and 2.73% (see table 8) compared with those of the E-rice, which shows that the method can promote the development of the whole growth cycle of the rice and finally obviously improve the rice yield.
TABLE 8 Effect of Amycolatopsis sp. MtRT-6 spore suspension on agronomic traits in rice
| Effective tillering number/number | Ear length/cm | Yield per gram of individual plant | Thousand grain weight/g | |
| E- | 5.23±0.236 | 21.153±0.237 | 19.53±0.897 | 32.25±0.286 |
| E+ | 7.72±0.321*** | 22.642±0.214*** | 31.60±1.288*** | 33.13±0.190** |
Indicates that the difference between E-and E + rice was significant (ANOVA, <0.001, <0.01), based on Tukey-Kramer test.
The results are combined to know that the growth promoting potential of the MtRT-6 is analyzed from physiological, biochemical and genome information, the amycolatopsis is applied to the leguminous plant medicago truncatula and the gramineae grain crop rice for the first time, and the growth promoting and yield increasing effects of the amycolatopsis on different families are verified.
The above examples are merely preferred embodiments of the present invention, and the scope of the present invention is not limited to the above examples. All technical schemes belonging to the idea of the invention belong to the protection scope of the invention. It should be noted that modifications and embellishments within the scope of the invention may be made by those skilled in the art without departing from the principle of the invention, and such modifications and embellishments should also be considered as within the scope of the invention.
Sequence listing
<110> university of Hunan
<120> rare actinomycetes having endogenic activity for promoting plant growth and use thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1425
<212> DNA
<213> endophytic rare Actinomycetes (Amycolatopsis sp. MtRT-6)
<400> 1
ggcgataggc cgtctacatg cagtcgacgc tgacgcacct tcggtgtgtg gatgagtggc 60
gaacgggtga gtaacacgtg ggtaatctgc cctgcactct gggataagcc ttggaaacga 120
ggtctaatac cggatatcac aatctctcgc atggggggtt gttgaaagtt ctggcggtgc 180
aggatgaacc cgcggcctat cagcttgttg gtggggtagt ggcctaccaa ggcgacgacg 240
ggtagccggc ctgagagggt gaccggccac actgggactg agacacggcc cagactccta 300
cgggaggcag cagtggggaa tattgcacaa tgggcgcaag cctgatgcag cgacgccgcg 360
tgagggatga cggccttcgg gttgtaaacc tctttcgcca gggacgaagc gcaagtgacg 420
gtacctggat aagaagcacc ggctaactac gtgccagcag ccgcggtaat acgtagggtg 480
cgagcgttgt ccggatttat tgggcgtaaa gagctcgtag gcggtttgtc gcgtcggccg 540
tgaaatctcc acgcttaacg tggagcgtgc ggtcgatacg ggcagacttg agttcggtag 600
gggagactgg aattcctggt gtagcggtga aatgcgcaga tatcaggagg aacaccggtg 660
gcgaaggcgg gtctctgggc cgatactgac gctgaggagc gaaagcgtgg ggagcgaaca 720
ggattagata ccctggtagt ccacgctgta aacgttgggc gctaggtgtg ggcgacatcc 780
acgttgtccg tgccgtagct aacgcattaa gcgccccgcc tggggagtac ggccgcaagg 840
ctaaaactca aaggaattga cggggggccc gcacaagcgg cggagcatgt ggattaattc 900
gatgcaacgc gaagaacctt acctgggctt gacatgcgcc agacatcccc agagatgggg 960
cttcccttgt ggttggtgta caggtggtgc atggctgtcg tcagctcgtg tcgtgagatg 1020
ttgggtttaa gtcccgcaac gagcgcaact ccttatccta cgttgccagc gacgttatgt 1080
cggggactcg tggggaagac tgccgggggg ttcaactcgg aggaaggtgg ggatgacgtc 1140
aagtcatcat gccccttatg tccagggctt cacacatgct acaatggctg gtacagaggg 1200
ctgcgatacc gcgaggtgga gcgaatccct taaagccggt ctcagttcgg atcgcagtct 1260
gcaactcgac tgcgtgaagt cggagtcgct agtaatcgca gatcagcaac gctgcggtga 1320
atacgttccc gggccttgta cacaccgccc gtcacgtcat gaaagtcggt aacacccgaa 1380
gcccatggcc caaccccgtc agggaggagt gtcgaactac tggcc 1425
Claims (10)
1. An endophytic rare actinomycete for promoting plant growth is characterized in that the endophytic rare actinomycete is Amycolatopsis sp.MtRT-6 and the strain preservation number is CGMCC No. 23411.
2. The rare actinomycete for endophyte according to claim 1, wherein the rare actinomycete for endophyte is obtained after isolation and purification from the root of alfalfa.
3. A rare endophyte spore suspension obtained by culturing the rare endophyte spore according to claim 1 or 2.
4. The rare endophyte actinomycete spore suspension of claim 3, wherein the concentration of the rare endophyte actinomycete spore suspension is 1 x 10 5 CFU/mL～1×10 8 CFU/mL。
5. Use of a suspension of endospores of a rare endophyte according to claim 3 or 4 to promote growth of a legume and/or a graminaceous plant.
6. Use according to claim 5, characterized in that it comprises the following steps: planting seedlings of leguminous plants and/or gramineous plants in soil, and irrigating an endophytic rare actinomycete spore suspension into the soil where roots of the leguminous plants and/or gramineous plants are located, and cultivating the seedlings of the leguminous plants and/or gramineous plants.
7. The use according to claim 6, wherein the suspension of endospores of rare endophyte actinomycete is infused in an amount of 1mL per strain; the concentration of the endophytic rare actinomycete spore suspension is 1 multiplied by 10 5 CFU/mL～1×10 7 CFU/mL; the leguminous plant is alfalfa; the gramineous plant is rice.
8. Use according to claim 5, characterized in that it comprises the following steps: embedding leguminous plants and/or gramineous plant seeds by using the endophytic rare actinomycetes spore suspension, and cultivating leguminous plants and/or gramineous plant seeds coated with the endophytic rare actinomycetes on the surface.
9. The use of claim 8, wherein prior to said embedding process, further comprising suspending spores of a rare-earth endophytic actinomyceteMixing the floating liquid with embedding agent; the proportion of the seeds of the leguminous plants and/or the gramineous plants, the embedding medium and the suspension of the spores of the endophytic rare actinomycetes is 1g to 463 mu L to 51 mu L; the embedding agent is xanthan gum; the volume fraction of the xanthan gum is 0.3%; the concentration of the endophytic rare actinomycete spore suspension is 1 multiplied by 10 6 CFU/mL～1×10 8 CFU/mL。
10. Use according to claim 9, wherein the seeds of leguminous plants are alfalfa seeds; the gramineous plant seed is a rice seed.
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