CN116640696A - Novel wheat rhizosphere soil Shulman's bacterium and application thereof - Google Patents

Abstract

The invention discloses a novel schumannellasp (schumannellasp.) M29 and application thereof. The novel strain M29 of the Shuman's fungus is separated from rhizosphere soil of winter wheat in Meuyu county in Hetian, can tolerate 3% NaCl, has the activities of dissolving indissoluble inorganic phosphorus and organic phosphorus, has urease and weaker activity of degrading cellulose, and has wide development and application prospects in the aspect of agricultural biofertilizer, and the strain can be applied to preparation of plant growth-promoting phosphorus-dissolving microbial agents, can reduce fertilizer consumption, promote plant growth and reduce fertilizer consumption.

Description

A new bacterium of Schulmania in wheat rhizosphere soil and its application

technical field

The invention belongs to the field of biological technology, and in particular relates to a new bacterium of Schumannella wheat rhizosphere and its isolation method and application.

Background technique

Phosphorus is one of the three major nutrient elements necessary for plant growth, and phosphorus in soil mainly exists in an insoluble state. For farmland ecosystems, applying phosphorus fertilizers to the soil is the main way of supplying phosphorus. However, existing studies have shown that the utilization rate of phosphorus fertilizers in the current season after being applied to the soil is 10-25%, and most of the others form insoluble phosphorus, such as calcium phosphate, iron phosphate, aluminum phosphate and other compounds.

Soil microorganisms drive the cycle of various elements in the soil. For example, some microorganisms can convert insoluble or immobilized phosphorus into forms that can be absorbed and utilized by plants, and these microorganisms are called Phosphate-solubilizing microorganisms (PSM). Compared with the rhizosphere and bare soil, the crop rhizosphere usually has a large number of phosphorus-dissolving microorganisms. Using its unique ecological function, people have isolated Bacillus, Burkholderia, Enterobacter and Erwinia from the soil. Bacteria capable of dissolving phosphorus. Among them, individual strains have been developed into growth-promoting phosphate-dissolving bacteria agents, and have been successfully applied in agricultural production.

At present, although there are many microbial strains with phosphorus-dissolving function in the existing technology, it is necessary to isolate and screen new and highly active plant rhizosphere phosphorus-dissolving bacteria, and to study its phosphorus-dissolving mechanism and its growth-promoting effect on plants. It is of great significance to reduce the amount of chemical fertilizers and ensure the green and sustainable development of agriculture.

Contents of the invention

Aiming at the low utilization rate of phosphate fertilizer, few highly active plant rhizosphere phosphorus-dissolving bacteria, and no literature and patent reports on the technical status of the application of Schumannela sp. in the preparation of growth-promoting phosphorus-dissolving bacteria agent, the present invention The invention aims at providing a new Schumannella sp. M29 and its application. The present invention isolates a new Schumannella sp. M29 from winter wheat rhizosphere soil in Moyu County, Hotan, Xinjiang. The bacterium can tolerate 3% NaCl and has the activity of dissolving insoluble inorganic phosphorus and organic phosphorus. , and has urease and weaker cellulose-degrading activity, the new strain Schumannella sp. (Schumannella sp.) M29 growth-promoting phosphate-dissolving bacteria agent can not only reduce the amount of chemical fertilizers, but also promote plant growth, and has broad development and application prospects in agricultural biological fertilizers.

In order to achieve the above technical effects, the present invention is realized through the following technical solutions.

The invention relates to a growth-promoting phosphate-dissolving bacterium agent of Schumannella sp. M29, which is prepared by fermentation of Schumannella sp. M29.

The new Schumannella sp. M29 provided by the present invention is screened and isolated from the rhizosphere soil of winter wheat in Moyu County, Hotan, Xinjiang. Through 16S rRNA sequence phylogenetic analysis and morphological analysis, the M29 strain belongs to Schumannella sp. Fungus. By sequencing the strain's gene, the obtained sequence was compared and analyzed by BLAST on the NCBI website. The 16SrRNA gene sequence of the strain M29 had the highest homology with Schumannella luteola KHIA (AB362159), with a similarity of 96.6%. Sequences with high homology were selected to construct 16S rRNA phylogenetic trees using different methods. Strain M29 was the closest to Schumannella luteola KHIA (AB362159), with confidence levels of 91, 73 and 70 respectively. After a series of multiphase taxonomic identification, it can be determined that The strain M29 is a new strain of Schumannella sp. and has typical characteristics of the new strain.

The Schumannella sp. M29 provided by the present invention has been identified at the molecular level by the above-mentioned well-known and recognized strain systems in the art, combined with identification and analysis based on morphological identification, physiological and biochemical characteristics of the multi-phase classification system, the Schumannella sp. M29 involved in the present invention There are several differences between Schumannella sp. M29 and the closest standard type strain of this genus. It is confirmed that this Schumannella sp. M29 belongs to a new species of Schumannella sp., and it is named It is Schumannella sp. M29, which has been preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures. The strain preservation number is CGMCC No. 24257, and the preservation date is January 06, 2022.

The gene sequence of the aforementioned Schumannella sp. M29 is shown in SEQ ID NO:1.

In the present invention, the isolation and activation medium of Schumannella sp. M29 is (g/L): glucose 0.5, yeast extract 0.5, peptone 0.5, acid hydrolyzed casein 0.5, soluble starch 0.5, pyruvate Sodium 0.3, dipotassium hydrogen phosphate 0.3, magnesium sulfate 0.05, agar 1, distilled water to 1000mL, pH 7.2.

In the present invention, the purification medium of Schumannella sp. M29 is: glucose 10.0, ammonium sulfate 0.5, yeast extract powder 0.5, NaCl 0.3, KCl 0.3, magnesium sulfate 0.3, ferrous sulfate 0.03, sulfuric acid Manganese 0.03, calcium phosphate 5.0, pH 7.0-7.5, agar 15.0.

Simultaneously, the present invention provides a kind of preparation method of Schumannella sp. M29 growth-promoting phosphate-dissolving bacterium agent, specifically comprises the following steps: the single bacterium colony of Schumannella sp. M29 Inoculated into NB culture medium, cultured for 2 days under the conditions of 30°C and 180rpm to obtain Schumannella sp. M29 seed solution; the Schumannella sp. M29 seed solution According to the inoculation amount of 1% by volume, it was inserted into the NB liquid medium, and fermented and cultivated at 30° C. and 180 rpm for 3 days to prepare the growth-promoting phosphate-dissolving agent of Schumannella sp. M29.

Further, the present invention provides an application of Schumannella sp. M29 in plant growth promotion, which can promote plant growth and has broad development and application prospects in plant growth promotion.

At the same time, the present invention provides the application of a new Schumannella sp. M29 growth-promoting phosphate-dissolving bacterium agent in plant growth promotion, which can promote plant growth and has broad development and application prospects in plant growth promotion .

Through the above technical scheme, the present invention obtains the following technical effects:

(1) The present invention provides a new Schumannella sp. M29, which has been identified at the molecular level by well-known and recognized strain systems in the art, combined with multi-phase classification based on morphological identification, physiological and biochemical characteristics, chemical characteristics, etc. Systematic identification analysis, Schumannella sp. M29 has many differences from the standard type strain with the closest homology of this genus, and it is indeed a new species of Schumannella sp. Because M29 belongs to a typical new strain, it is necessary to carry out the preservation according to the legal requirements.

(2) The present invention provides a new Schumannella sp. (Schumannella sp.) M29 which can tolerate 3% NaCl, has the activity of dissolving insoluble organic phosphorus and inorganic phosphorus, and can dissolve calcium phytate, calcium phosphate, etc. in the soil. A kind of insoluble phosphorus compound; it can prepare plant growth-promoting phosphorus-dissolving bacteria agent, which has broad development and application prospects. The new Schumanella sp. M29 growth-promoting phosphorus-dissolving bacteria agent can not only reduce the amount of chemical fertilizers, but also It can promote plant growth and has broad development and application prospects in agricultural biological fertilizers.

Description of drawings

Figure 1 shows the phylogenetic tree of strain M29 constructed by the neighbor-joining method based on the 16S rRNA gene sequence.

Fig. 2 shows the phylogenetic tree of strain M29 constructed by using the maximum parsimony method based on the 16S rRNA gene sequence.

Figure 3 shows the phylogenetic tree of the strain M29 constructed by the maximum likelihood method based on the 16S rRNA gene sequence.

Figure 4 shows the morphological characteristics of strain M29.

Detailed ways

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the following examples. All the raw and auxiliary materials selected in the present invention, as well as the selected bacterial strain cultivation methods are well known in the art and selected, and the % involved in the present invention is volume percentage, unless otherwise specified.

The NB medium used in the preparation of Schumannella sp. M29 growth-promoting phosphate-dissolving bacteria agent of the present invention is (g/L): peptone 10, beef extract powder 3, sodium chloride 5, deionized water 1L, pH 7.4±0.2.

Example 1: Isolation, purification and identification of Schumannella sp. M29

(1) Separation and purification

The soil was collected from the rhizosphere of winter wheat in the Hotan area of Xinjiang Uygur Autonomous Region by the plate dilution method, put into a plastic bag, sealed and brought back to the laboratory, and stored at 4°C until use. Accurately weigh 2g of soil sample, add ^{18mL of} sterile water, shake on a shaker ^at 30° ^C and ^180rpm for 30min; 10 ^-6 , 10 ^-7 gradient, take 200 μL of sample suspension on the bacterial inorganic phosphorus medium plate, and incubate at 30°C for 7 days. Pick a single colony, streak and purify it on the same medium, and use 20% skimmed milk powder to freeze and cryopreserve the obtained pure bacteria.

(2) 16S rRNA gene identification

1. Extraction of DNA

Schumannella sp. M29 was inoculated in purified liquid medium and cultured at 30°C for 3 days to obtain bacterial cells. Genomic DNA was extracted using a bacterial genomic DNA rapid extraction kit.

2. PCR amplification

16S rRNA sequence primers:

27F: 5'-AGA GTT TGATCC TGG CTC-3';

1492R: 5'-CGG CTACCT TGT TAC GAC TT-3'.

Reaction system and conditions:

3. Sequencing

The PCR amplification product was detected by electrophoresis and purified and then sequenced. The sequence length is 1514bp. The sequence result is shown in SEQ ID No: 1. The BLAST homologous sequence search was carried out at NCBI, and the MEGA 7.0 software commonly used in this field was used to establish through the Neighbor-Joining method. Phylogenetic tree (repeated sampling 1000 times), the results are shown in Figures 1-3, the resulting sequence was compared and analyzed on the NCBI website, and it was found that the 16S rRNA gene sequence of the bacterial strain M29 was identical to that of the Schumanella Schumannellaluteola KHIA (AB362159). The origin is the highest, with a similarity of 96.6%, see Table 1 for specific data. In the phylogenetic tree constructed by different methods based on the 16S rRNA gene sequence, the 16S rRNA sequence of the strain M29 has the closest relationship with the Schumannellaluteola KHIA (AB362159) strain, and the strain M29 and S. branches, the confidence levels are 91, 73 and 70 respectively; it indicates that the support rate of the strain M29 as a new strain is extremely high, and it shows excellent stability in the evolutionary tree, through the synthesis of the similarity and homology of the strains It was determined that the acquired strain numbered M29 in the genus Schumannella belonged to a typical new strain of the genus Schumannella.

Table 1: Homology of the strains of the present invention

Example 2: Taxonomic identification of Schumannella sp. M29

(1) Colony morphology of Schumannella sp. M29

The strain M29 to be observed was inoculated on the NB plate, and cultured at 30°C for 4 days. After the colony covered the entire plate, the characteristics of the colony were observed, recorded and photographed, and the scanning electron micrograph of the colony was recorded at the same time. The recorded results are shown in Figure 4.

It can be seen from the results in Figure 4 that the strain M29 was cultured in NB medium at 30°C for 4 days, and the diameter of the colony was about 1mm. About 0.8-0.9 μm.

Based on the above biological characteristics, the above strain M29 was identified as Schumannella sp. The strain has been deposited in the Budapest Treaty Microorganism International Depository Unit: China Microbiological Culture Collection Management Committee General Microbiology Center (CGMCC), address: China. Beijing. No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, postal code: 100101, Deposit date: January 06, 2022, and the deposit number is CGMCC No.24257.

(2) Physiological and biochemical characteristics of Schumannella sp. M29

The strain M29 was inoculated on NB medium, and the physiological and biochemical characteristics were detected respectively. Characteristic analysis of growth temperature, salinity, pH range, gelatin liquefaction, nitrate reduction, hydrogen peroxide production, urease, amylase, esterase, cellulose decomposition, carbon source utilization, etc. mainly refer to the "Common Bacterial System Identification Manual" .

Strain M29 grew better on TSA and NB differential media, but poorer on NA and R ₂ A differential media. It can grow on the medium containing 0-3% NaCl, the optimum concentration is 0%; it can grow at 25-40°C, and the optimum growth temperature is 30°C; it can grow at pH 6-9, and the optimum pH is 7.0. Positive for milk coagulation, negative for gelatin liquefaction, negative for milk peptonization; positive for urease, positive for hydrolysis of Tween 20 and 80, weak starch hydrolysis activity; no degradation of cellulose; able to utilize glucose, sucrose, lactose, raffinose, D -Xylose, rhamnose, acetic acid, melibiose, mannitol, galactose, sweet alcohol, oxalic acid, malic acid, sorbitol, ribose, unavailable mannose, cellobiose, arabinose, succinic acid, lemon Acids; maltose, trehalose, cellobiose, lactose, mannose, glycerin, aspartic acid, glutamic acid, histidine. Can utilize cysteine, glutamic acid, glutamine, L-tryptophan, L-aspartic acid, xanthine, hydroxyproline, glycine, adenine, L-arginine, L-color Acid, Histidine, Leucine, Lysine, L-Alanine.

Using plate chromogenic method, the strain M29 was spotted on the phosphate bacteria medium containing insoluble lecithin or calcium phosphate, and after culturing at 30°C for 3 days, an obvious transparent circle was observed around the colony, indicating that the strain M29 has a difficulty in dissolution. Soluble organic phosphorus and inorganic phosphorus activity.

It can be seen that the strain M29 has similar physiological and biochemical characteristics to the closest homologous strain of this genus, Schumannella luteola KHIA (AB362159), but there are many differences, and it is a potential new species of Schumanella.

Based on comparative analysis of 16S rRNA gene sequence homology, morphological identification, and analysis of physiological and biochemical characteristics, it is clearly different from the standard type strain with the closest homology, and it is determined that the strain M29 is a new species of Schumania. According to its source of isolation, it was named Schumannella sp. M29.

Embodiment 3: the preparation of Schumannella sp. (Schumannella sp.) M29 growth-promoting phosphate-dissolving bacteria agent

Present embodiment is on the basis of embodiment 1-2, and the preparation method of Schumannella sp. .) A single colony of M29 was inoculated into NB medium, and cultured at 30°C and 180rpm for 2 days to obtain a seed solution of Schumannella sp. M29; the new Schumannella sp. .) M29 seed liquid, according to the inoculation amount of 1% by volume, was inserted into the NB liquid medium, and fermented and cultivated at 30°C and 180rpm for 3 days, and the new Schumannella sp. M29 growth-promoting solution was prepared. Phosphorus agent.

Example 4: Application of Schumannella sp. M29

Referring to the method described in the standard NY/T 1847-2010, inoculate M29 into Erlenmeyer flasks containing 10.0g/L calcium phosphate, aluminum phosphate, iron phosphate, lecithin or calcium phytate respectively, and shake it Bed 200rpm, 30 ℃ culture 72h. After the end, the content of available phosphorus in the supernatant was measured, and the specific test results are shown in Table 2. Among them, phosphate-dissolving liquid medium: glucose 0.5g, yeast extract 0.5g, peptone 0.5g, acid hydrolyzed casein 0.5g, soluble starch 0.5g, sodium pyruvate 0.3g, dipotassium hydrogen phosphate 0.3g, magnesium sulfate 0.05g , agar 15g, distilled water to 1000mL, pH 7.2.

Table 2: Phosphorus-solubilizing activity of strain M29

As can be seen from the data in Table 2, the bacterial strain M29 provided by the invention has a lytic activity of 7.37mg/L to calcium phosphate, a lytic activity to aluminum phosphate of 4.03mg/L, and a lytic activity to iron phosphate of 7.92mg/L; The dissolving activity of calcium carbonate is 29.04mg/L, and the dissolving activity to lecithin is 0.52mg/L; This shows that the bacterial strain M29 provided by the invention has significant activity of dissolving insoluble organic phosphorus and inorganic phosphorus.

Inoculate the strain M29 into the activation medium, and culture it in a shaker flask at 30°C for 2 days to obtain the activated bacterial liquid of Schumannella tritici M29; prepare the phosphate-dissolving liquid medium; The ratio was inserted into the phosphate-dissolving liquid medium, and cultured at 180 rpm at 30°C for 3 days. Molybdenum-antimony anti-colorimetric method was used to measure the phosphorus-dissolving ability of the phosphorus-dissolving and growth-promoting bacteria agent, and the M29 growth-promoting phosphorus-dissolving bacteria agent was inoculated into 10.0g/L calcium phosphate, aluminum phosphate, iron phosphate, lecithin or calcium phytate respectively. In the Erlenmeyer flask of the phosphorus bacteria liquid culture medium, set the shaker at 200rpm, 30 ℃ for 72h. After the end, measure the content of available phosphorus in the supernatant, and the specific results are shown in attached table 3. Among them, phosphate-dissolving liquid medium: glucose 0.5g, yeast extract 0.5g, peptone 0.5g, acid hydrolyzed casein 0.5g, soluble starch 0.5g, sodium pyruvate 0.3g, dipotassium hydrogen phosphate 0.3g, magnesium sulfate 0.05g , agar 15g, distilled water to 1000mL, pH=7.2.

Table 3: Phosphorus-dissolving activity of strain M29 growth-promoting phosphate-dissolving bacteria agent

As can be seen from the data in Table 3, the bacterial strain M29 growth-promoting phosphate-dissolving bacteria agent provided by the invention has a dissolving activity of 7.59 mg/L to calcium phosphate, a dissolving activity of 4.27 mg/L to aluminum phosphate, and a dissolving activity of 8.16 mg/L to iron phosphate. mg/L; the dissolving activity to calcium phytate is 31.21mg/L, and the dissolving activity to lecithin is 0.65mg/L; this shows that the bacterial strain M29 growth-promoting phosphate-dissolving bacteria agent provided by the invention has significant dissolving insoluble organic Phosphorus and inorganic phosphorus activity.

The above data analysis shows that adopting the Schumanella new bacterium (Schumannella sp.) M29 provided by the invention can tolerate 3% NaCl, dissolve the insoluble phosphorus in the soil, especially the organophosphorus calcium phytate, can increase to a certain extent The content of soluble phosphorus in the soil can promote the absorption and utilization of phosphorus by plants, thereby promoting plant growth and reducing the amount of phosphorus fertilizer used. It can be applied to the preparation of growth-promoting phosphorus-dissolving bacteria agent, which can not only reduce the amount of chemical fertilizer, but also It promotes plant growth, has good ecological and social benefits, and has broad development and application prospects in agricultural biological fertilizers.

Claims (6)

1. A novel strain of schumann bacteria (schumannellasp.) M29, wherein the novel strain of schumann bacteria (schumannellasp.) M29 is deposited in the chinese microbiological bacterial deposit management committee common microbiological centre, with a bacterial deposit number of: cgmccno.24257.

2. A new strain of schumann bacteria (schumannellasp.) M29 according to claim 1, wherein the new strain of schumann bacteria (schumannellasp.) M29 has the gene sequence set forth in SEQ ID NO: 1.

3. A novel schumannellasp (schumannellasp.) M29 growth-promoting phosphorus-dissolving microbial inoculum is characterized in that the growth-promoting phosphorus-dissolving microbial inoculum is obtained by fermenting schumannellasp (schumannellasp.) M29.

4. A process for the preparation of a new schumannella (schumannellasp.) M29 growth-promoting lysophosphate according to claim 3, characterized in that it comprises the following steps: inoculating a single colony of a strain of schumannellasp (schumannellasp.) M29 into NB culture medium, and culturing for 2 days at 30 ℃ in a shaking bottle to obtain a schumannellasp (schumannellasp.) M29 seed solution; the schumann bacteria (schumannellasp.) M29 seed solution is inoculated into NB liquid culture medium according to the inoculation amount of 1% of the volume ratio, and is fermented and cultured for 3 days at 30 ℃ and 180rpm, so as to obtain the schumannellasp (schumannellasp.) M29 growth-promoting phosphorus-dissolving bacterial agent.

5. Use of a novel schumannella sp.m 29 according to claim 1 for plant growth promotion.

6. Use of a new schumannella sp (schumannellasp.) M29 growth-promoting phosphorus-solubilizing microbial agent according to claim 3 for plant growth promotion.