A kind of saccharotrichum with soil improving function and its application

Abstract

The present invention discloses a saccharothricin fungus with the function of improving soil and its application. Saccharothrix sp. ) named Saccharothrix sp. ML-D1, deposit number: CCTCC NO: M20241588, deposited in China Center for Type Culture Collection on July 17, 2024, deposited at Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The present invention obtains a strain with phosphorus and potassium solubilizing function through multiple rounds of screening, and the strain can greatly increase the content of available phosphorus and available potassium in the soil; at the same time, it can increase exchangeable calcium, reduce soil exchangeable acid and effective manganese content, and improve soil nutrient effectiveness and buffering capacity. The strain and the bacterial agent prepared by the strain have high agricultural application prospects and economic value.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a saccharotrichum with the function of improving soil and an application thereof. Background Art

[0002] With the rapid development of modern agriculture, the problems of soil degradation and fertility decline have become increasingly prominent, seriously restricting the sustainability of agricultural production. Soil degradation not only affects the yield and quality of crops, but may also lead to the deterioration of the ecological environment. Although traditional chemical fertilizers can quickly improve the short-term fertility of soil, long-term excessive use will destroy the natural balance of the soil, causing soil structure damage, decreased organic matter content, reduced beneficial microbial populations, and even soil salinization and heavy metal accumulation, which in turn affects the growth environment of crops and food safety. In order to cope with these problems, people have begun to seek more environmentally friendly and sustainable soil management methods. Microbial fertilizers have received increasing attention because they can promote the balance of soil ecosystems and improve the natural fertility of soil. The active microorganisms contained in microbial fertilizers can improve the physical, chemical and biological properties of the soil through their life activities, improve the water retention and aeration of the soil, and increase the number and diversity of beneficial microorganisms in the soil, thereby improving the fertility of the soil and the growth conditions of crops.

[0003] Plant rhizosphere growth-promoting bacteria can improve plant growth, increase soil fertility, increase yields, and reduce pathogens or abiotic stresses. In existing reports, many plant rhizosphere growth-promoting bacteria have been reported to have good growth-promoting and soil-improving effects. However, despite these potential advantages, rhizosphere growth-promoting bacteria still face some challenges in practical applications. For example, there are problems such as unstable activity, poor adaptability, and inconsistent performance under different soil types and environmental conditions. These problems limit its widespread application in soil improvement and agricultural production.

[0004] At present, the resources of bacterial strains with strong stability and outstanding effects that can be used in agricultural production are still relatively scarce. There are no reports on Saccharotrichum with nutrient activation and plant growth promotion functions and its application in soil improvement and fertility enhancement. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a novel Saccharotrichum strain, which is obtained by a specific screening method and has the characteristics of improving soil quality and promoting plant growth. The present invention also aims to provide a method of using the strain to improve soil fertility and crop yield.

[0006] The present invention provides a saccharothricin fungus having the function of improving soil Saccharothrix sp. ), named Saccharothrix sp.ML-D1, deposit number: CCTCC NO: M20241588, was deposited in the China Center for Type Culture Collection on July 17, 2024, and the deposit address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] The second aspect of the present invention also provides a microbial agent containing the Saccharothrix.

[0008] Preferably, the bacterial agent is a liquid preparation, and the preparation contains Saccharotrichum Saccharothrix sp. The concentration of ML-D1 was 1×10 7 ~1×10 9 CFU / mL.

[0009] The third aspect of the present invention further provides the use of the Saccharotrichum or the microbial agent in improving soil.

[0010] As a further description of the above scheme: the simple method for preparing the microbial agent for improving soil is as follows:

[0011] Saccharotrichum Saccharothrix sp. ML-D1 was activated and cultured in NA solid medium at 29°C-31°C for 20h-28h by streaking method. Then, a single colony was picked with a sterile inoculation loop and inoculated into NA liquid medium. The culture was shaken at 33°C-37°C and 220r / min-230r / min for 36h-50h to obtain the fermentation broth. The fermentation broth was prepared into 1.8×10 8 -2.5×10 8 CFU / mL of Saccharothrix Saccharothrix sp. ML-D1 bacterial solution is reserved;

[0012] The activated solid culture medium comprises the following components: 2.8-3.2 g beef extract, 9.5-10.5 g peptone, 4.5-5.5 g sodium chloride, 14-16 g agar powder, and pH 7.2-7.5 per 1000 mL sterilized distilled water.

[0013] Saccharothrix Saccharothrix sp. The usage of ML-D1 microbial agent is 8-15kg / mu, diluted 50-100 times with water.

[0014] In a fourth aspect, the present invention further provides the use of the Saccharotrichum or the microbial agent in promoting plant growth. Experiments have shown that the microbial agent can significantly improve the yield and quality of potatoes and bok choy.

[0015] Compared with the prior art, the present invention has the following beneficial effects: the present invention obtains a strain with the function of solubilizing phosphorus and potassium and promoting growth through multiple rounds of screening, and the strain can greatly improve the effectiveness of phosphorus and potassium nutrients in the soil; at the same time, it can significantly increase the exchangeable calcium, reduce the exchangeable acid and effective manganese content of the soil, eliminate the acidic soil barrier factors and improve the buffering capacity of the soil. Furthermore, according to the records of the embodiments, the present invention can increase the yield of crops and improve the quality of crops, and enhance the market competitiveness of agricultural products. The saccharotrichum of the present invention is easy to culture and apply on a large scale, is simple to operate, is easy to be accepted and applied by agricultural producers, and has high practicality. The strain and its preparation have high agricultural application prospects and economic value.

[0016] The strain deposit information is as follows:

[0017] Category naming: Saccharothrix sp. ML-D1;

[0018] Deposit number: CCTCC NO: M20241588;

[0019] Deposit date: July 17, 2024;

[0020] Depository: China Center for Type Culture Collection;

[0021] Storage address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Saccharothricin Saccharothrix sp. Colony morphology of ML-D1;

[0023] Figure 2 Saccharothricin Saccharothrix sp. ML-D1 cell morphology under electron microscope;

[0024] Figure 3 Saccharothricin Saccharothrix sp. Phylogenetic tree between ML-D1 and reference strains;

[0025] Figure 4 Saccharothricin Saccharothrix sp. Phosphorus (organic phosphorus) solubilization effect diagram of ML-D1;

[0026] Figure 5 Saccharothricin Saccharothrix sp. Phosphorus dissolution (inorganic phosphorus) effect diagram of ML-D1;

[0027] Figure 6 Saccharothricin Saccharothrix sp. Graph showing the growth-promoting effect of ML-D1. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solution. In the following embodiments, the materials, reagents, etc. used were obtained from commercial sources unless otherwise specified.

[0029] Example 1 Saccharotrichum Saccharothrix sp. Screening and morphological characteristics of ML-D1

[0030] (1) Soil samples from the roots of corn were collected from the Jiuxian Town of Malong, Qujing, Yunnan. After removing obvious stones and tree roots from the soil in an ultra-clean workbench, 2.0 g of soil sample was weighed and added to 200 mL of sterile water containing glass beads. The soil sample was then placed in a constant temperature air bath shaking incubator (30°C, 180 r / min) for 4–6 hours to fully break up the soil sample, release and mix the bacteria contained in the soil sample. This was 10 -2 The soil dilution solution was diluted in equal proportions to prepare a concentration gradient of 10 -3 , 10 -4 , 10 -5 The soil dilution was inoculated with 10 -3 , 10 -4 , 10 -5 200 μL of each soil dilution was applied evenly on a solid medium for organophosphorus-dissolving bacteria (10.0 g glucose, 0.5 g ammonium sulfate, 0.5 g yeast extract powder, 0.3 g sodium chloride, 0.3 g potassium chloride, 0.3 g magnesium sulfate, 0.03 g ferrous sulfate, 0.03 g manganese sulfate, 0.2 g lecithin, 1.0 g calcium carbonate, 20.0 g agar powder, pH 7.2) using a sterile applicator. Each concentration was repeated 3 times. After marking, the plates were inverted in a 30°C biochemical incubator for 3-4 days of incubation. The growth of the colonies and the formation of transparent circles were closely observed. After the incubation, colonies with obvious transparent circles were picked from the plates with an inoculation needle, and separated by streaking on a beef extract peptone solid medium to obtain single colonies. After marking, the plates were inverted in a 30°C biochemical incubator for 3 days. This operation was repeated 1-2 times. After incubation, individual strains with inconsistent colony morphology were observed and selected for purification to obtain pure culture strains.

[0031] (2) Pick the above single colony and transfer it to potassium dissolving screening medium (sucrose 10.0 g, potassium dihydrogen phosphate 1.0 g, ammonium sulfate 0.5 g, magnesium sulfate heptahydrate 1 g, yeast powder 0.2 g, sodium chloride 0.1 g, calcium carbonate 0.1 g, ferric chloride 0.005 g, feldspar powder 5.0 g, agar 15.0-20.0 g, pH 7.2), and culture at 28°C, 190 rpm for 30 hours. Select a single colony to obtain strain ML-D1, which has the function of dissolving phosphorus and dissolving potassium.

[0032] 2. Morphological identification and physiological and biochemical characteristics of strain ML-D1

[0033] The strain ML-D1 was streaked on the culture medium and cultured at 28°C for 24 hours. The colony characteristics such as colony shape, transparency and colony color were observed. Figure 1 and Figure 2 The colony morphology of strain ML-D1 is as follows: the bacteria are earthy yellow, round, with complete edges, dull, dry, and opaque; its physiological and biochemical characteristics are: Gram-negative, aerobic, with an optimal growth temperature of 28-37°C, an optimal growth pH of 7.0-8.0, an optimal salt concentration of 1-3%, and has the characteristics of dissolving phosphorus and potassium.

[0034] 3. Molecular Biological Identification of Strain ML-D1

[0035] The DNA of strain ML-D1 was extracted using the Ezup column bacterial genomic DNA extraction kit. The 16S rRNA sequence of the strain was amplified by PCR using universal primers 27F and 1492R. The 16S rDNA of the strain was amplified using universal primers (commissioned to Beijing Qingke Biotechnology Co., Ltd. for synthesis): 27F (upstream primer, sequence 5'-AGTTTGATCMTGGCTCAG-3', SEQ ID NO.2) and 1492R (downstream primer, sequence 5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.3). The amplified product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were corrected and spliced ​​using Vector NTI Advance 11 software, and the spliced ​​sequences were searched for homologous sequences using BLASTn to determine their taxonomic status. The 16S r RNA gene sequence of strain ML-D1 is shown in SEQ ID NO.1.

[0036] Homologous sequence search results showed that strain ML-D1 and Saccharothrips ( Saccharothrix sp. ) reached 99.8%. Twelve 16SrDNA sequences with relatively high homology were selected as reference objects, and the phylogenetic tree between strain ML-D1 and the reference strain was constructed using the Neighbour-Joining method and Mega 7 software. Figure 3 As shown. Combining morphological, physiological and biochemical characteristics with molecular sequences, the strain ML-D1 was identified as Saccharotrichum ( Saccharothrix sp. )。 The Saccharotrichum ( Saccharothrix sp. ) was deposited in China Center for Type Culture Collection on July 17, 2024 and named Saccharothrix sp. ML-D1, accession number: M20241588.

[0037] Example 2 Analysis of the Phosphate Solubilization Effect of Strain ML-D1

[0038] Use an inoculation loop to pick colonies on the solid culture medium of organophosphate-solubilizing bacteria, spot four identical colonies on each plate, mark them, and invert them in a 30°C biochemical incubator for 2 days. Use the cross method to determine the diameter of the phosphate-solubilizing circle (D) and the diameter of the colony (d), and calculate their respective average values. The ratio of the two (D / d) is used to preliminarily judge the phosphate-solubilizing ability of the organophosphate-solubilizing strain, and a solubility index (D / d) analysis is performed. The qualitative determination of inorganic phosphate-solubilizing bacteria is the same as above, except that the solid culture medium of organophosphate-solubilizing bacteria is replaced with a solid culture medium of inorganic phosphate-solubilizing bacteria. The results showed that after culturing the strain ML-D1 on the organophosphate solid culture medium for 2 days, an obvious degradation transparent zone appeared around the colonies. As the culture continued, the degradation zone increased significantly, indicating that the strain ML-D1 can dissolve organic and inorganic phosphorus and has strong phosphate-solubilizing properties (Table 1, Table 2, Figure 4 and Figure 5 ).

[0039] Table 1 Analysis of the phosphate solubilization of strain ML-D1 on organic phosphorus

[0040]

[0041] Table 2 Analysis of the solubility of inorganic phosphorus by strain ML-D1

[0042]

[0043] The composition of the above-mentioned organophosphorus solid culture medium is: 10.0 g of glucose, 0.5 g of ammonium sulfate, 0.5 g of yeast extract powder, 0.3 g of sodium chloride, 0.3 g of potassium chloride, 0.3 g of magnesium sulfate, 0.03 g of ferrous sulfate, 0.03 g of manganese sulfate, 0.2 g of lecithin, 1.0 g of calcium carbonate, 20.0 g of agar powder, pH 7.0~7.5, per 1000 mL of distilled water.

[0044] The components and concentrations of the inorganic phosphorus-dissolving solid culture medium are as follows: 10.0 g of glucose, 0.5 g of ammonium sulfate, 0.3 g of sodium chloride, 0.3 g of potassium chloride, 0.3 g of magnesium sulfate, 0.03 g of ferrous sulfate, 0.03 g of manganese sulfate, 5.0 g of tricalcium phosphate, 20.0 g of agar powder, and pH 7.2-7.4 per 1000 mL of distilled water.

[0045] The phosphorus solubility rate is the effect of strain ML-D1 on the release of insoluble phosphorus in the fermentation broth after inoculation in the phosphorus-containing fermentation broth. The content of phosphate in the bacterial fermentation broth is determined by the molybdenum antimony colorimetric method and calculated. Phosphorus solubility rate calculation: Phosphorus solubility rate = (phosphate content in the fermentation broth mg / L-phosphate content in the control solution mg / L) / (phosphate ester concentration (in terms of phosphate) mg / L-phosphate content in the control solution mg / L) × 100%. The results showed that the phosphate content in both the organic phosphorus and inorganic phosphorus fermentation broths in the experimental group increased significantly compared with the control group, and the phosphorus solubility rate of strain ML-D1 was in the range of 19.78-39.16%.

[0046] Table 3 Phosphorus increment results in fermentation broth

[0047]

[0048] The isolated and purified single bacterial colony was inoculated into a potassium-solubilizing liquid medium (agar was not added to the potassium-solubilizing screening medium), with potassium feldspar powder as the only potassium source, and cultured in a shaking incubator at 30°C and 130 r / min for 48 h. The K in the potassium-solubilizing bacteria fermentation broth was detected by flame photometer. + The potassium dissolving rate was calculated by comparing the content with that of the control group without potassium dissolving bacteria. The results showed that strain ML-D1 can significantly improve the potassium dissolving efficiency.

[0049] Table 4 Potassium increment results in fermentation broth

[0050]

[0051] Example 3 Potato growth promotion experiment with strain ML-D1

[0052] Trial Location

[0053] Qujing Hui Ze is located at N26°00′34″, E103°24′14″, 2470 meters above sea level. The test site is flat, the soil type is red soil, and the soil fertility is medium and uniform. Test variety: Yunshu 921. Before planting, the soil samples of the arable layer were collected and five conventional soil items were analyzed.

[0054] Data Collection

[0055] Soil and plant sample collection

[0056] Using the 5-point "S" type mixing method, collect about 1kg of soil samples, divide the samples into 2 parts, and air-dry one part in the cool place of the laboratory. After removing impurities, measure soil pH, organic matter, available potassium, soil hydrolyzable nitrogen, soil total phosphorus, soil total potassium, cation exchange capacity, and exchangeable calcium. When the potatoes are mature and harvested, randomly collect plant samples to be tested in the production area of ​​each plot, evenly select plant samples, dry them, weigh them and measure the cumulative amount of dry matter, and measure the total nitrogen, total phosphorus and total potassium content. Calculate the cumulative amount of nitrogen, phosphorus and potassium absorption; calculation formula: nutrient absorption (kg / mu) = biomass (kg / mu) × nutrient content (%). How to use the microbial agent of Saccharothrips: 10kg / mu, dilute 50-100 times with water, and then irrigate.

[0057] The preparation method of the microbial inoculant of Saccharotrichum is as follows:

[0058] (1) Selection of bacterial strains: Saccharotrichum ML-D1.

[0059] (2) Preparation of Saccharothricin ML-D1 inoculum: Activate Saccharothricin D1 stored in ultra-low temperature (-80℃) glycerol: Use the streak method to culture at 30℃ for 24 h using NA solid medium. Then pick a single colony with a sterile inoculation loop and inoculate it into NA liquid medium. Cultivate at 35℃, 220 r / min for 2 days to obtain the fermentation liquid of the strain. Use sterile water to prepare the fermentation liquid of the strain to 2×10 8 CFU / mL of Saccharothrix ML-D1 bacterial agent is ready for use.

[0060] The components of the activated solid culture medium are as follows: 3.0 g beef extract, 10.0 g peptone, 5.0 g sodium chloride, 15 g agar powder per 1000 mL sterile distilled water, pH 7.4.

[0061] Experimental treatment design

[0062] Potato planting was carried out according to a randomized block design, and the treatments were as follows:

[0063] CK: customary fertilization, compound fertilizer (NPK 15-15-15) 50 kg / mu, as control;

[0064] T1: customary fertilization, compound fertilizer (NPK 15-15-15) 50kg / mu + ML-D1 bacterial agent 10kg / mu.

[0065] Field cultivation management

[0066] Determination of economic traits of potato

[0067] Indicators include: yield per mu. The potato yield is determined after the potatoes mature. Three points are collected for each treatment. The protection row is removed from each point. The yield is measured manually in the middle of the harvest, and the commercial quality of the tubers in each treatment is observed and recorded. Determination of potato biological yield and dry matter content: A 5-point sampling method is used for each treatment, and 2 potato plants are taken from each point, for a total of 10 plants. The fresh weight of the aboveground part and roots of potatoes, and the fresh weight of potato tubers are measured separately. Cut them into pieces manually and put them into bags, put them in a forced air drying oven at 105°C; kill them for 30 minutes, and bake them at 80°C until constant weight. The dry matter weight of potato tubers is weighed, and quality indicators such as vitamin C, starch and protein are measured.

[0068] Results and Analysis

[0069] Table 5 Fresh weight and yield of potato tubers under different treatments (kg)

[0070]

[0071] Note: In the table, the lowercase letters in the results of multiple comparison analysis of variance indicate that the different treatments have reached significant differences at the p<0.05 level, the same as in the following table.

[0072] The results in Table 5 show that the ML-D1 bacterial agent of Saccharotrichum can significantly increase the weight of single-plant large potatoes, total weight and yield per mu.

[0073] Effects of microbial inoculants on potato quality

[0074] Table 6 Potato quality under different treatments

[0075]

[0076] The test results show that the ML-D1 bacterial agent can significantly increase the starch and protein content of potatoes and improve the quality of potatoes.

[0077] Table 7 Nitrogen, phosphorus and potassium absorption of potatoes under different treatments (kg / mu)

[0078]

[0079] Table 7 shows that the nitrogen, phosphorus and potassium absorption of potatoes treated with the ML-D1 bacterial agent of Saccharotrichum saccharotrichum was significantly higher than that of the conventional fertilization group.

[0080] The results of soil improvement by the Saccharotrichum ML-D1 inoculant are shown in Tables 8 and 9.

[0081] Table 8 Physical and chemical properties of soils under different treatments

[0082]

[0083] Table 9 Physical and chemical properties of soils under different treatments

[0084]

[0085] It can be seen from the above table that the ML-D1 bacterial agent of Saccharotrichum significantly increased the soil available phosphorus, fast-acting potassium, and exchangeable calcium, reduced the soil exchangeable acid and available manganese content, and improved the soil nutrient effectiveness and buffering capacity.

[0086] Example 4: Growth promotion of Saccharotrichum saccharotrichum on Boehmeria boehmeria

[0087] The test site is located in Zhuangke Village, Songhuaba, Panlong District, Kunming City, Yunnan Province, in the south-central part of Yunnan Province, at 25°13′17″N, 102°52′11″E, and 1994 m above sea level. The test land type is water-converted dry land, the soil texture is medium loam, and the soil fertility is relatively high.

[0088] Experimental treatment

[0089] The experiment was conducted in three treatments, each with three replicates, for a total of nine plots, using a randomized block design. The plot area was 1.8 m × 20 m = 36 m 2 The treatments are as follows:

[0090] CK: blank treatment; M1: pure organic fertilizer alone; M2: pure organic fertilizer alone + Saccharothricin ML-D1 inoculant;

[0091] Except for the control, the application rate of organic fertilizer in each test plot was 10.8 kg, and the application rate of Saccharotrichum ML-D1 inoculant was 0.54 kg.

[0092] Experimental Results

[0093] Table 10 Effects of different fertilization treatments on plant height of Shanghai green

[0094]

[0095] As shown in Table 10, the plant height of each fertilization treatment was higher than that of the control group, but did not reach a significant level; the SPAD values ​​of the fertilization treatments were significantly higher than those of the control treatment CK. In terms of yield, the yield of each fertilization treatment was higher than that of the unfertilized treatment CK. There were significant differences between treatments M1 and M2 and treatment CK, which were 9.63% and 14.28% higher than the control treatment CK, respectively. Among them, the yield of M2 treatment was the highest, and the yield increase was significant compared with the control treatment CK. This shows that the application of Saccharotrichum ML-D1 inoculant can improve the yield and quality of Shanghai green.

[0096] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, but rather, the present invention extends to all other methods and applications with the same functions.

Claims

1. A strain of Saccharotrichum with soil-improving function ( Saccharothrix sp. ), characterized in that The Saccharotrichum Saccharothrix sp. ML-D1, deposit number: CCTCC NO: M20241588, was deposited in the China Center for Type Culture Collection on July 17, 2024, and the deposit address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. A microbial agent containing the Saccharotrichum according to claim 1.

3. Use of the Saccharotrichum according to claim 1 or the microbial agent according to claim 2 in improving soil.

4. Use of the Saccharotrichum according to claim 1 or the microbial agent according to claim 2 in improving soil, characterized in that: The operation is as follows: Saccharotrichum Saccharothrix sp. ML-D1 was activated and cultured in NA solid medium at 29°C-31°C for 20h-28h by streaking method. Then, a single colony was picked with a sterile inoculation loop and inoculated into NA liquid medium. The culture was shaken at 33°C-37°C and 220r / min-230r / min for 36h-50h to obtain the fermentation broth. The fermentation broth was prepared into 1.8×10 8 -2.5×10 8 CFU / mL of Saccharothrix Saccharothrix sp. ML-D1 bacterial solution is reserved; Saccharothrix Saccharothrix sp. The usage of ML-D1 bacterial solution is 8-15kg / mu, diluted 50-100 times with water.

5. The use according to claim 4, characterized in that: The components of the NA solid culture medium used for the activation are as follows: 2.8-3.2 g beef extract, 9.5-10.5 g peptone, 4.5-5.5 g sodium chloride and 14-16 g agar powder per 1000 mL sterilized distilled water, pH 7.2-7.

5.

6. Use of the Saccharotrichum according to claim 1 or the microbial agent according to claim 2 in promoting the growth of potatoes or bok choy.

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