CN110846262B

CN110846262B - Serratia marcescens SZ201 and application thereof

Info

Publication number: CN110846262B
Application number: CN201911376584.1A
Authority: CN
Inventors: 陈强
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2020-08-14
Anticipated expiration: 2039-12-27
Also published as: CN110846262A

Abstract

The invention discloses a Serratia marcescens SZ201 and application thereof, wherein the Serratia marcescens (Serratia marcocens) SZ201 is preserved in China center for type culture collection (CCTCC NO) in 8.2.2019, and the preservation number is as follows: m2019597, deposit organization address: the serratia marcescens SZ201 is applied to flue-cured tobacco production, can effectively improve potassium absorption of flue-cured tobacco plants and leaves, reduces potassium reflux of plants after topping, is favorable for improving the quality of tobacco leaves, and therefore, is used as an excellent production strain of a special bio-organic fertilizer for flue-cured tobacco, and has a good application prospect.

Description

Serratia marcescens SZ201 and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to serratia marcescens SZ201 and application thereof.

Background

Tobacco (Nicotiana tabacum L) is an important economic crop in China, plays an important role in national economic development, and is also one of important characteristic industries for increasing income of tobacco growers in mountainous areas and causing poverty to be rich in rural areas. In 2018, the total amount of industrial and commercial tax profits is 11500 hundred million yuan in the tobacco industry in China. The tobacco leaves are not only raw materials of cigarettes, but also a large number of natural chemical substances such as nicotine, solanesol, nicotine and the like contained in the tobacco leaves, but also high-quality chemical and medical raw materials, so that the problem of how to stabilize and improve the quality of flue-cured tobacco is still urgently solved.

Potassium is one of three essential nutrient elements for plant growth and development, has important functions in the growth, development and metabolic processes of plants, can be used as an activator of various enzymes, enhances the utilization of nitrogen by the plants, promotes the synthesis and transportation of assimilates such as proteins and saccharides in the plants, participates in cell permeation regulation, enhances the photosynthesis of the plants, enhances the stress resistance of the plants, and is closely related to the quality of crops. The potassium deficiency of the plants can lead to slow growth of crops, yellow or burnt leaves along the leaf edges, and incomplete root system development.

Potassium is the element with the largest growth demand of tobacco plants, and is usually 1.4 times of the nitrogen demand and 3.5 times of the phosphorus demand. In addition, potassium is a recognized tobacco quality element and plays an important role in improving the usability of tobacco leaves. Internationally, the average potassium content of high-quality tobacco leaves is more than 2 percent, while the average potassium content of tobacco leaves in most tobacco areas in China is 1.8 percent and is lower than the international standard of the potassium content of high-quality tobacco leaves. Therefore, the low content of potassium in the tobacco leaves becomes an important factor for restricting the tobacco quality in China, especially the tobacco quality. Researches find that under the influence of the nutrient absorption characteristics of flue-cured tobacco products, the potassium content in most of tobacco leaves of flue-cured tobacco varieties after the tobacco leaves are subjected to topping shows a greatly reduced trend, and the potassium content is an important factor influencing the improvement of the quality of flue-cured tobacco. The common counter measures in production are to select the potassium-rich genotype flue-cured tobacco variety and to apply chemical potash fertilizer in later period. However, if the fertilizer is excessively applied for a long time, the production cost is increased, the soil structure is deteriorated, the soil microbial community structure and functional diversity are damaged, and non-point source pollution is caused due to nutrient loss. Therefore, a plan of 'double reduction of pesticide and fertilizer' is proposed in 2016 agricultural rural areas; in 2017, a scheme of developing a fruit, vegetable and tea organic fertilizer to replace a chemical fertilizer is implemented. The organic fertilizer and the biological organic fertilizer are widely popularized to be used, and the using amount of the fertilizer is reduced, so that the method is one of important contents in the national thirteen-five planning.

Serratia marcescens (also called as "Ganoderma lucidum") produces fresh red pigment, widely distributed in nature, and is a resident flora in water and soil. Researches show that the serratia marcescens has various biological activities and is more and more valued by researchers. After searching Chinese patent public networks, the content of 32 invention patents related to Serratia marcescens has been granted by the national patent office in recent years, and 33 patent patents are in a state of being examined actually, the contents of the invention patents mainly focus on the aspects that the Serratia marcescens is used for fermenting and producing the Lin-Bacin, researching materials in molecular biology, researching and developing anticancer drugs by fermentation liquor, antagonistic bacteria of crop pathogens, growth-promoting bacteria of crops, degrading bacteria of organic pollutants, repairing bacteria of soil heavy metals and the like, but the invention does not see the use of the Serratia marcescens for producing biological organic fertilizers, reducing potassium reflux of flue-cured tobacco plants, promoting potassium absorption of flue-cured tobacco plants, improving the potassium content of flue-cured tobacco leaves and improving the quality of flue-cured tobacco.

Disclosure of Invention

Aiming at the technical problem of low potassium content in flue-cured tobacco leaves in China, the invention provides the serratia marcescens SZ201 which has an excellent effect of promoting the absorption of potassium in the flue-cured tobacco leaves and can effectively reduce the potassium reflux of flue-cured tobacco plants after topping, so that the invention also provides the application of the serratia marcescens in improving the potassium content in the flue-cured tobacco leaves, and can promote the potassium content in the flue-cured tobacco leaves to be improved, thereby improving the quality of the tobacco leaves.

The invention is realized by the following technical scheme:

the method collects wheat straws for a returning test from a rice field of a modern agricultural research and development base of alder spring town Sichuan agricultural university in Chongzhou, Sichuan province, and obtains serratia marcescens SZ201 through separation and purification. Serratia marcescens SZ201 is small rod-shaped, gram-negative, non-spore, flagellar and non-capsule. Culturing for 48 hours on a beef extract peptone culture medium, wherein the bacterial colony is red, the character is circular or irregular, the diameter of the bacterial colony is 2-3 mm, the edge is neat, and the surface is wet and smooth.

Extracting the total DNA of the strain SZ201, amplifying a 16S rDNA fragment, and determining a 16S sequence shown in SEQ ID NO. 1. The measured 16S sequence was compared with the National Center for Biotechnology Information (NCBI) database in the United states, and SZ201 was compared with Serratia marcescens (Serratia marcescens subsp. marcescens) ATCC13880 in GenBank^TThe similarity of SZ201 was the highest (99.6%), thereby confirming that SZ201 belongs to Serratia marcescens (Serratiamarcessensis subsp. marccessens) in classification.

Based on the characteristics, the invention provides a serratia marcescens SZ201, and the classification and the naming of the serratia marcescens SZ201 are as follows: serratia marcescens, with the Latin chemical name: serratia marcocens are already preserved in the China center for type culture Collection in 2019, 8 and 2, and the preservation number is CCTCC NO: m2019597, deposit organization address: wuhan university No. 299 in eight ways in Wuhan district, Wuhan city, Hubei province.

The Serratia marcescens (SZ 201) obtained by the invention is applied to flue-cured tobacco production, can improve the potassium content of flue-cured tobacco leaves, and particularly can reduce the potassium reflux amount in flue-cured tobacco plants after the flue-cured tobacco topping period.

The serratia marcescens SZ201 microbial inoculum or the culture solution thereof is applied to flue-cured tobacco production, so that the potassium content of flue-cured tobacco leaves is improved, and the invention is also within the protection scope.

In addition, the invention also comprises a biological preparation for improving the content of potassium in flue-cured tobacco leaves by using the serratia marcescens SZ201, wherein the biological preparation contains serratia marcescens SZ201 powder or culture solution thereof, and the biological preparation can be mixed with organic fertilizer to prepare microbial fertilizer and the like.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the invention provides a Serratia marcescens (Serratia marcocens) SZ201 for producing a biological organic fertilizer, which is applied to flue-cured tobacco production, wherein the potassium content of upper and middle leaves of flue-cured tobacco is respectively increased by 6.71% and 11.83%. Meanwhile, the potassium reflux of leaves and roots of different parts of flue-cured tobacco is reduced by 9.81-24.06%, and meanwhile, the Serratia marcescens Serratia marcescens SZ201 has stronger capability of improving the effectiveness of soil nutrients, can be used as an excellent strain for agricultural production, and has good application prospect.

Drawings

FIG. 1 shows the colony morphology of Serratia marcescens SZ201 cultured on beef extract peptone medium.

FIG. 2 shows the shape of the cells observed by a microscope with Serratia marcescens SZ 201.

FIG. 3 is a phylogenetic diagram of the 16S rDNA sequence of Serratia marcescens SZ 201.

Detailed Description

The objects, technical solutions and advantageous effects of the present invention will be described in further detail below.

It is to be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention claimed, and unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Example 1:

this example relates to the isolation, purification and preservation of Serratia SZ 201.

Collecting samples: collecting a part of decomposed wheat straw samples in a straw returning experiment from a paddy field of a modern agriculture research and development base of Alnus Town agricultural university in Chongzhou city of Sichuan province, storing at a low temperature and bringing the samples back to a laboratory.

Preparing a culture medium: the beef extract peptone comprises 3 g of beef extract, 10 g of peptone, 5 g of sodium chloride, 20g of agar, pH7.0-7.2 and 1000 mL of water.

Separating and purifying strains: taking wheatAdding 5.0 g of rice soil sample with straw returned to the field into 45 mL of sterile water, and carrying out shake culture for 2 h at 28 ℃ and 140 r/min; sucking 1mL of sample liquid, preparing bacterial suspension by adopting a gradient method, and diluting to 10%^-510 of 1mL each^-3、10^-4And 10^-5Plating the bacterial suspension on a beef extract peptone culture medium plate, and culturing at 28 ℃ for 2-3 d. After the bacteria grow out, selecting bacterial colonies with different forms from the plate, streaking the bacterial colonies on a beef extract peptone plate, culturing the bacterial colonies for 1 to 2 days at the temperature of 28 ℃, selecting single bacterial colonies for simple staining or gram staining, and combining microscopic observation until obtaining a pure bacterial strain with the serial number of SZ 201. The purified strain is inoculated into a test tube slant culture medium of beef extract peptone culture medium and stored at 4 ℃.

As shown in FIG. 1, the colony morphology of the strain SZ201 obtained by separation and purification in this example after culturing for 48 h on a beef extract peptone medium plate is circular, red (i.e., the dark part in the figure), 1-2 mm in diameter, neat in edge, and flat and wet. FIG. 2 shows the microscopic shape of the SZ201, which is small rod-shaped, gram-negative, non-spore, flagellated, and non-capsule.

Example 2:

this example relates to the 16S rDNA amplification sequencing and phylogenetic analysis of Serratia marcescens SZ201

Extracting total DNA of Serratia marcescens SZ201 strain, amplifying 16S segment by using the total DNA as a template and 27F and 1492R as primers, and performing PCR reaction by using a Bio-RADMCyclerTM instrument.

Reaction system (50 μ l): mu.l of 2 XPCRMix 25. mu.l each of primers 27F and 1492R (10. mu.M), 1. mu.l of DNA template, and made up to 50. mu.l with ultrapure water; the nucleotide sequences of primers 27F and 1492R are shown as SEQID number 2 and SEQID number 3.

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C for 1 min, annealing at 54 deg.C for 1 min, extension at 72 deg.C for 2min, and circulation for 30 times; final extension at 72 ℃ for 8 min.

The PCR amplification product was detected on 1.0% agarose gel electrophoresis and then sent to Shanghai Bioengineering Co., Ltd for sequence determination. The software DNAMAN7.0 is used for calculating the similarity of gene sequences. The sequencing result is shown as SEQ ID No. 1.

The obtained sequence results were compared in the National Center for Biotechnology Information (NCBI) database, and the model strain having the highest similarity of the genus Raynaud was selected as the reference strain, and a phylogenetic tree was constructed using MEGA 7.02 Neighbor-joining method (Neighbor-joining), as shown in FIG. 3, with a self-expansion value (bootstrap) of 1000. And the 16S rDNA sequence of SZ201 is combined with a model strain Serratia marcescens (Serratia marcescens subsp. marcescens) ATCC13880^TThe similarity was 99.6%.

Based on the above characteristics, the strain SZ201 was identified as Serratia marcescens (Serratia marcocen). The strain is stored in China center for type culture Collection in 2018, 8, 2 and the preservation number is CCTCC NO: m2019597.

Example 3:

the embodiment relates to a method for preparing a biological organic fertilizer by using serratia marcescens SZ 201.

Inoculating serratia marcescens SZ201 to a beef extract peptone liquid culture medium for activated culture, culturing at 28 ℃ to logarithmic phase, adding bacterial liquid into an organic fertilizer according to 5% of the weight of the organic fertilizer when the strain SZ201 is cultured to exponential phase, and fully and uniformly mixing the organic fertilizer with the bacterial liquid to prepare the serratia marcescens biological organic fertilizer. The number of viable bacteria in the biological organic fertilizer is more than 0.2 hundred million/g.

Example 4

The embodiment relates to an effect experiment of Serratia marcescens SZ201 on improving the content of potassium in flue-cured tobacco leaves

The test soil is collected from the test soil collected from double forest villages (E104 degrees 20 '38' and N30 degrees 30 '11') of Longquay tea shop of city, Sichuan province, the soil type is neutral purple soil, and the initial fertility characteristic is as follows: 44.08mg/kg of alkaline hydrolysis nitrogen, 8.55mg/kg of quick-acting phosphorus, 66.01mg/kg of quick-acting potassium and 1.85% of organic matters, and is applied to a university Chengdu district experimental greenhouse of Sichuan agriculture university. The pot experiment is provided with two treatments of T1 and T2, wherein, the T1 is applied with organic fertilizer which is equal to the treatment of T2, and the T2 is applied with SZ201 biological organic fertilizer. The inoculation mode was SZ201 bio-organic fertilizer hole application, with the bio-organic fertilizer prepared in example 3 applied. The method comprises the following specific steps: filling the soil to be testedUniformly mixing, weighing and potting (6 kg/pot), applying organic fertilizer and bio-organic fertilizer according to treatment, keeping the environment ventilated and sufficient light during the growth period of the flue-cured tobacco, controlling the water content of soil to be 20-30%, and using water for irrigation as dechlorinated water. Collecting root, stem and leaf samples of flue-cured tobacco plants in the topping stage and the mature stage, deactivating enzyme at 105 deg.C, oven drying at 80 deg.C to constant weight, pulverizing, sieving with 35 mesh sieve, and sieving with H₂SO₄-H₂O₂The total potassium content (%) of different parts of the plant is measured by a digestion-flame photometer method, and the potassium reflux rate of the flue-cured tobacco plant is calculated.

The increase rate of the potassium content of the flue-cured tobacco plants is% = (the potassium content of T2-the potassium content of T1)/the potassium content of T1 is 100%

Flue-cured tobacco plant potassium reflux rate% = (top-removed period potassium content-mature period potassium content)/top-removed period potassium content 100%

The results in Table 1 show that the potassium content in the upper and middle leaves of the maturation period is increased by 6.71% and 11.83% in the T2 treatment compared to the T1 treatment. Further analyzing the change of the potassium reflux rate of different parts of the flue-cured tobacco plants after different treatment topping periods, compared with T1, the potassium reflux rate of different parts of leaves of flue-cured tobacco is obviously reduced by T2 treatment. Wherein, the potassium reflux rate of the upper leaves of the flue-cured tobacco plants is reduced by 11.32 percent, and the reduction amplitude is maximum; the return rate of potassium in the roots and the stalks is reduced by 7.86 percent and 5.73 percent respectively.

TABLE 1 Potassium content in different parts of different treatments at different times

Note: different treatments at the same site were analyzed for significance, with differences in lower case letters within the same column indicating significant differences (p < 0.05).

Example 5:

this example relates to the use of Serratia marcescens SZ201 to increase the available nutrient content of soil.

In the tobacco pot experiment, a bacterium-free control (T1) and a serratia SZ201 (T2) are designed and inoculated, and the change conditions of the test strains on the contents of slow-acting potassium and fast-acting phosphorus in soil in different growth periods of flue-cured tobacco plants are researched. Respectively on the root of a Chinese grollCollecting flue-cured tobacco rhizosphere soil in different treatment stages, a topping stage and a mature stage, drying a soil sample, removing impurities, air-drying, grinding and sieving, wherein the content of slow-release potassium in the soil adopts HNO₃The quick-acting phosphorus content of the soil is determined by adopting a boiling method and adopting NaHCO₃And (4) measuring by an extraction method.

The results in table 2 show that the T2 treatment can obviously improve the contents of soil slow-release potassium and available phosphorus in different growth periods of flue-cured tobacco. After the inoculation of SZ201, the slow-release potassium content of the soil in the flue-cured tobacco in the bulk stage and the mature stage is respectively increased by 4.05 percent and 2.61 percent, the available phosphorus content of the soil is respectively increased by 17.50 percent and 14.71 percent, and the utilization efficiency of soil nutrients is effectively improved.

TABLE 2 variation of content of slow-acting potassium and fast-acting phosphorus in different treated soils

Note: the difference in lower case letters within the same column between different treatments at the same time period means a significant difference (p < 0.05).

From the above examples 1-5, it can be known that, aiming at the technical problem of low potassium content in flue-cured tobacco leaves in China, the invention obtains a Serratia marcescens (Serratia marcescens) SZ201 by separating and purifying from rice soil with wheat straw returned to the field, the Serratia marcescens SZ201 is applied to flue-cured tobacco production, and has the effect of improving the potassium content in the flue-cured tobacco leaves, and particularly, aiming at a main cultivated variety Yunyan 87 of flue-cured tobacco in Sichuan province, the SZ201 biological organic fertilizer is applied, so that the potassium content of the upper and middle leaves of the flue-cured tobacco can be obviously improved, and the potassium reflux quantity of the flue-cured tobacco roots, stems and different parts of the flue-cured tobacco leaves after topping can. Certainly, the serratia marcescens SZ201 can also be used as a biological agent when being applied to flue-cured tobacco production. In addition, the invention also relates to the application of the serratia marcescens SZ201 in improving the available nutrients of tobacco-planting soil, and tests prove that the slow-release content and the available phosphorus content of the tobacco-planting soil inoculated with the serratia marcescens SZ201 are increased, and the tobacco-planting soil has a certain promotion effect on improving the available nutrients of the tobacco-planting soil.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and all simple modifications and equivalent variations of the above embodiments according to the technical spirit of the present invention are included in the scope of the present invention.

Sequence listing

<110> Sichuan university of agriculture

<120> Serratia SZ201 and application thereof

<160>3

<170>SIPOSequenceListing 1.0

<210>1

<211>1326

<212>DNA

<213>（Serratia marcescens）

<400>1

GCTTGCTCCC TGGGTGACGA GCGGCGGACG GGTGAGTAAT GTCTGGGAAA CTGCCTGATG 60

GAGGGGGATA ACTACTGGAA ACGGTAGCTA ATACCGCATA ACGTCGCAAG ACCAAAGAGG 120

GGGACCTTCG GGCCTCTTGC CATCAGATGT GCCCAGATGG GATTAGCTAG TAGGTGGGGT 180

AATGGCTCAC CTAGGCGACG ATCCCTAGCT GGTCTGAGAG GATGACCAGC CACACTGGAA 240

CTGAGACACG GTCCAGACTC CTACGGGAGG CAGCAGTGGG GAATATTGCA CAATGGGCGC 300

AAGCCTGATG CAGCCATGCC GCGTGTGTGA AGAAGGCCTT CGGGTTGTAA AGCACTTTCA 360

GCGAGGAGGA AGGTGGTGAG CTTAATACGC TCATCAATTG ACGTTACTCG CAGAAGAAGC 420

ACCGGCTAAC TCCGTGCCAG CAGCCGCGGT AATACGGAGG GTGCAAGCGT TAATCGGAAT 480

TACTGGGCGT AAAGCGCACG CAGGCGGTTT GTTAAGTCAG ATGTGAAATC CCCGGGCTCA 540

ACCTGGGAAC TGCATTTGAA ACTGGCAAGC TAGAGTCTCG TAGAGGGGGG TAGAATTCCA 600

GGTGTAGCGG TGAAATGCGT AGAGATCTGG AGGAATACCG GTGGCGAAGG CGGCCCCCTG 660

GACGAAGACT GACGCTCAGG TGCGAAAGCG TGGGGAGCAA ACAGGATTAG ATACCCTGGT 720

AGTCCACGCT GTAAACGATG TCGATTTGGA GGTTGTGCCC TTGAGGCGTG GCTTCCGGAG 780

CTAACGCGTT AAATCGACCG CCTGGGGAGT ACGGCCGCAA GGTTAAAACT CAAATGAATT 840

GACGGGGGCC CGCACAAGCG GTGGAGCATG TGGTTTAATT CGATGCAACG CGAAGAACCT 900

TACCTACTCT TGACATCCAG AGAACTTAGC AGAGATGCTT TGGTGCCTTC GGGAACTCTG 960

AGACAGGTGC TGCATGGCTG TCGTCAGCTC GTGTTGTGAA ATGTTGGGTT AAGTCCCGCA 1020

ACGAGCGCAA CCCTTATCCT TTGTTGCCAG CGGTTCGGCC GGGAACTCAA AGGAGACTGC 1080

CAGTGATAAA CTGGAGGAAG GTGGGGATGA CGTCAAGTCA TCATGGCCCT TACGAGTAGG 1140

GCTACACACG TGCTACAATG GCGTATACAA AGAGAAGCGA CCTCGCGAGA GCAAGCGGAC 1200

CTCATAAAGT ACGTCGTAGT CCGGATTGGA GTCTGCAACT CGACTCCATG AAGTCGGAAT 1260

CGCTAGTAAT CGTAGATCAG AATGCTACGG TGAATACGTT CCCGGGCCTT GTACACACCG 1320

CCCGTC 1326

<210>2

<211>19

<212>DNA

<213> Artificial sequence (27F)

<400>2

AGAGTTGATC CTGGCTCAG 19

<210>3

<211>20

<212>DNA

<213> Artificial sequence (1492R)

<400>3

CGGTTACCTT GTTACGACTT 20

Claims

1. A serratia marcescens SZ201 is characterized in that: the classification of Serratia marcescens SZ201 is named as: serratia marcescens, with the Latin chemical name: serratia marcocens are already preserved in the China center for type culture Collection in 2019, 8 and 2, and the preservation number is CCTCC NO: m2019597, deposit organization address: wuhan university No. 299 in eight ways in Wuhan district, Wuhan city, Hubei province.

2. The Serratia marcescens SZ201 according to claim 1, wherein: the 16S rDNA sequence of the Serratia marcescens SZ201 is shown in SEQ ID NO. 1.

3. The Serratia marcescens SZ201 according to claim 1, wherein: serratia marcescens SZ201 is small rod-shaped, gram-negative, non-spore, flagellar and non-capsule.

4. The Serratia marcescens SZ201 according to claim 1, wherein: the serratia marcescens SZ201 is cultured on a beef extract peptone culture medium for 48 hours, the bacterial colony is red, the character is circular or irregular, the diameter of the bacterial colony is 2-3 mm, the edge is neat, and the surface is wet and smooth.

5. The use of the serratia marcescens SZ201 as claimed in claim 1, wherein: the serratia marcescens SZ201 is applied to flue-cured tobacco production, and the potassium content of flue-cured tobacco leaves is improved.

6. The use of the serratia marcescens SZ201 as claimed in claim 1, wherein: the serratia marcescens SZ201 microbial inoculum or the culture solution thereof is applied to flue-cured tobacco production, so that the potassium content of the flue-cured tobacco leaves is improved.

7. The use of the serratia marcescens SZ201 as claimed in claim 1, wherein: the serratia marcescens SZ201 is used for preparing a biological preparation for improving the potassium content of flue-cured tobacco leaves, and the biological preparation contains serratia marcescens SZ201 powder or culture solution thereof.