CN110669703B

CN110669703B - Pseudomonas putida S9 and application thereof in promoting growth of coprinus comatus

Info

Publication number: CN110669703B
Application number: CN201911129441.0A
Authority: CN
Inventors: 李冠喜; 李秋月; 于颖媛; 张璐; 张鹤露; 亓超凡; 吴克兢; 郭帅
Original assignee: Qufu Normal University
Current assignee: Qufu Normal University
Priority date: 2019-11-18
Filing date: 2019-11-18
Publication date: 2022-02-22
Anticipated expiration: 2039-11-18
Also published as: CN110669703A

Abstract

The invention discloses Pseudomonas putida S9 and application thereof in promoting growth of coprinus comatus, relating to the technical field of edible fungi, wherein the Pseudomonas putida is classified and named as Pseudomonas putida (Pseudomonas putida) S9, is preserved in China Center for Type Culture Collection (CCTCC) No. M2019635; the preservation date is as follows: 8, 8 and 16 in 2019; and (4) storage address: wuhan university in Wuhan, China. The growth promoter is applied to promoting the growth of coprinus comatus, the growth speed of coprinus comatus hyphae added with pseudomonas putida S9 is accelerated, the hyphae are thick and dense, the stipe of the sporocarp is long, the yield of the sporocarp is increased, the growth rate of the coprinus comatus can be effectively improved, the production cycle of the coprinus comatus is shortened, the yield of the coprinus comatus can be improved, the green, healthy and pollution-free products are ensured, the consumption of auxiliary materials is reduced, and the production economy and social benefits of edible fungi are improved.

Description

Pseudomonas putida S9 and application thereof in promoting growth of coprinus comatus

Technical Field

The invention relates to the field of edible fungus biotechnology, and in particular relates to pseudomonas putida S9 and application thereof in promoting growth of coprinus comatus.

Background

Coprinus comatus is named as Coprinus comatus, is named after being similar to chicken shreds in meat quality because of the shape of the Coprinus comatus, has no chicken flavor, is a rare fungus product with commercial potential developed manually in recent years, and is known as 'new in fungus'. The coprinus comatus is rich in nutrition, delicious in taste, excellent in mouthfeel and high in nutritional value. The fruiting body is large, the pileus is cylindrical, when the pileus is opened for 40 minutes, the inner edge pileus is melted into liquid in the shape of ink juice, and the stipe becomes slender. The diameter of the pileus is 3-5 cm, the height is 9-11 cm, the surface brown color is changed into light brown color, and the pileus is broken into larger scales along with the growth of the pileus. The mushroom flesh is white, the stipe is white, the mushroom is cylindrical and is relatively long and thin, the mushroom is gradually thick downwards, the length of the mushroom is 7-25 cm, the thickness of the mushroom is 1-2 cm, and the mushroom is smooth. The traditional coprinus comatus planting method has low yield and more chemical pollution, and can not meet the requirements of consumers. The microorganism is vital to the growth of the plant, can decompose organic substances in soil to form humus and release nutrients, maintain the normal growth of plant cells, can convert soil carbon and fix inorganic nutrient elements, increases the utilization rate of the soil and achieves the effect of continuous culture. Therefore, the method is especially important for overcoming the defects of the traditional technology, reducing the use of chemical fertilizers and searching for suitable microorganisms, and the method can not cause human health hazards and environmental pollution while promoting the increase of the yield of the coprinus comatus.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide pseudomonas putida S9 which has the function of promoting the growth of coprinus comatus; the invention aims to solve the technical problem of providing the application of pseudomonas putida S9 in promoting the growth of coprinus comatus so as to solve the problems of low yield and high chemical pollution of the traditional coprinus comatus planting method.

In order to solve the problems, the invention adopts the technical scheme that:

pseudomonas putida, which is classified and named as Pseudomonas putida (Pseudomonas putida) S9, has been preserved in China center for type culture Collection with the preservation number of CCTCC No. M2019635; the preservation date is as follows: 8, 8 and 16 in 2019; and (4) storage address: wuhan university in Wuhan, China.

Application of pseudomonas putida S9 in promoting growth of coprinus comatus.

The application comprises the following steps: bacterial liquid culture, soil covering cultivation and fruiting management.

The pseudomonas putida S9 is sprayed in a fermentation liquor mode before soil covering cultivation.

Further, the concentration of the pseudomonas putida S9 in the fermentation liquor is 1.5X 10⁹cfu/mL～2.0×10⁹cfu/mL。

Further, the spraying amount of the fermentation liquor of the pseudomonas putida S9 is 500mL/m²。

Has the advantages that: compared with the prior art, the pseudomonas putida S9 is screened and separated from the coprinus comatus mushroom stick cultivation material and identified, the pseudomonas putida S9 is applied to promoting the growth of coprinus comatus, the growth speed of coprinus comatus hyphae added with pseudomonas putida S9 is accelerated, the hyphae are dense and thick through bacterial liquid culture, soil covering cultivation and fruiting management, the coprinus comatus hyphae are long in stipe and the fruiting body yield is increased, the growth rate of the coprinus comatus can be effectively increased, the production period of the coprinus comatus is shortened, the coprinus comatus yield can be increased, the production economy and social benefits of edible fungi are improved, the growth rate of the coprinus comatus is effectively increased, the green, healthy and pollution-free products are guaranteed, the auxiliary material consumption is reduced, and the production economy and social benefits of the edible fungi are improved.

Drawings

FIG. 1 is a graph showing the results of co-culturing Pseudomonas putida S9 with a Coprinus comatus fungus on a plate for 5 days;

FIG. 2 is a graph showing the effect of Pseudomonas putida S9 on the diameter of coprinus comatus hyphae in a dish;

FIG. 3 is a graph showing the effect of Pseudomonas putida S9 on the height of the stipe of a coprinus comatus fruiting body;

FIG. 4 is a graph showing the effect of Pseudomonas putida S9 on the bioconversion rate of Coprinus comatus fruiting body;

FIG. 5 is a graph showing the effect of Pseudomonas putida S9 on the yield per square meter of Coprinus comatus;

FIG. 6 is a phylogenetic tree diagram of Pseudomonas putida S9.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to be limiting.

Example 1

1) Isolation screening and identification of bacteria

The coprinus comatus mushroom stick cultivation material is randomly adopted from Chenyu fungus industry development limited company in Jinxiang county, diluted and coated by a rich culture medium enrichment flat plate, and 42 bacterial isolates are obtained by separation from the coprinus comatus mushroom stick cultivation material. Then separating and purifying by using an NA culture medium.

Wherein the formula of the NA culture medium comprises 10g of peptone, 5g of beef extract, 5g of sodium chloride, 20g of agar and pH7.2-7.4.

Inoculating the strains which are separated and purified by primary screening and stored into a conical flask of NA liquid culture medium, culturing for 48h at 28 ℃ at 180r/min in a shaking table to obtain cultured saturated bacterial liquid, inoculating 0.2mL of the bacterial liquid into a rich culture medium plate (20 g of glucose, 200g of potato, 10g of peptone, 3g of yeast extract, 15-20g of agar, 1000mL of water and pH7.0) respectively according to the requirement of aseptic operation by using a liquid transfer gun, uniformly coating by using a coater, taking 1cm of coprinus comatus fungus by using a puncher, and placing the coprinus comatus fungus in the center of the plate coated with the fungus. And adding 0.2mL of sterile water into the control group, inoculating the control group, placing the control group in an incubator, culturing at 28 ℃, observing the growth condition of hyphae every day, and photographing and recording. The result of the 5d culture is shown in figure 1, the influence of the screened bacteria on the coprinus comatus hypha diameter is shown in figure 2, the hypha radius of the co-culture group is obviously higher than that of the control group, and the growth promoting rate is gradually increased.

The 16S rDNA sequence of the strain is amplified, the product after PCR amplification is sent to Shanghai biological engineering company to complete sequence determination, the sequence is shown as SEQ ID NO.1, the sequencing result is subjected to Blast analysis on NCBI, and a phylogenetic tree is constructed by utilizing MEGA7.0, which is shown as figure 6. The sequencing result is aligned by Blast, and the strain is identified as Pseudomonas putida (Pseudomonas putida) S9.

3) Physiological and biochemical characteristics

Performing biological characteristic observation of the pseudomonas putida S9 by adopting gram staining; performing biochemical characteristic determination on the screened pseudomonas putida S9 by using methods such as citrate utilization experiment, indole experiment, gelatin liquefaction, Vop (VP) experiment and the like; results are shown in table 1:

TABLE 1 physio-biochemical characteristics of Pseudomonas putida S9

Name of item	Results	Name of item	Results
				Gram stain	Negative of	MR experiment	Negative of
Hydrogen peroxide	Positive for	Indole experiments	Negative of
				Vp experiment	Negative of	Citric acid salt	Positive for
Liquefaction of gelatin	Negative of	Starch hydrolysis	Negative of

Example 2

Effect of Pseudomonas putida S9 on fruiting

In a school cooperation edible fungus enterprise workshop, a planting area of 200 square meters is selected, the growth environment, the ingredient formula, the fermentation, bagging and fungus growing are consistent with the standardized production, wherein the concentration of 100 square covering soil is 1.5 multiplied by 10 per square meter before inoculation⁹cfu/mL～2.0×10⁹500mL of the bacterial liquid of the pseudomonas putida S9 cfu/mL, and sterile water with the same total volume added by 100 square of soil covering is used as a control to observe the fruiting speed and the yield of the coprinus comatus. The effect of pseudomonas putida S9 on the fruiting body traits of the coprinus comatus strains is shown in fig. 3-5, fig. 3 showing that the average stem height of the added bacterial coprinus comatus is 2cm higher than the average stem height of the coprinus comatus; FIG. 4 shows a 10% increase in bioconversion of Coprinus comatus with added bacteria; FIG. 5 shows that the yield of the coprinus comatus fruiting body added with bacteria is increased by about 1kg per square meter; the average fresh weight of single plants in the co-culture group is increased, the stipe is long, the full bag time is shortened, and the total yield and the biotransformation rate of single fruiting bodies are increased.

Sequence listing

<110> university of Qufu Master

<120> Pseudomonas putida S9 and application thereof in promoting growth of coprinus comatus

<130> 1

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1421

<212> DNA

<213> Pseudomonas putida

<400> 1

acacatgcaa agtcgagcgg atgagaagag cttgctcttc gattcagcgg cggacgggtg 60

agtaatacct aggaatctgc ctggtagtgg gggacaacgt ttcgaaagga acgctaatac 120

cgcatacgtc ctacgggaga aagcagggga ccttcgggcc ttgcgctatc agatgagcct 180

aggtcggatt agctagttgg tgaggtaatg gctcaccaag gctacgatcc gtaactggtc 240

tgagaggatg atcagtcaca ctggaactga gacacggtcc agactcctac gggaggcagc 300

agtggggaat attggacaat gggcgaaagc ctgatccagc catgccgcgt gtgtgaagaa 360

ggtcttcgga ttgtaaagca ctttaagttg ggaggaaggg cagtaagcga ataccttgct 420

gttttgacgt taccgacaga ataagcaccg gctaactctg tgccagcagc cgcggtaata 480

cagagggtgc aagcgttaat cggaattact gggcgtaaag cgcgcgtagg tggttcgtta 540

agttggatgt gaaatccccg ggctcaacct gggaactgca tccaaaactg gcgagctaga 600

gtagggcaga gggtggtgga atttcctgtg tagcggtgaa atgcgtagat ataggaagga 660

acaccagtgg cgaaggcgac cacctgggct catactgaca ctgaggtgcg aaagcgtggg 720

gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgtcaa ctagccgttg 780

gaatccttga gattttagtg gcgcagctaa cgcattaagt tgaccgcctg gggagtacgg 840

ccgcaaggtt aaaactcaaa tgaattgacg ggggcccgca caagcggtgg agcatgtggt 900

ttaattcgaa gcaacgcgaa gaaccttacc aggccttgac atccaatgaa ctttccagag 960

atggattggt gccttcggga acattgagac aggtgctgca tggctgtcgt cagctcgtgt 1020

cgtgagatgt tgggttaagt cccgtaacga gcgcaaccct tgtccttagt taccagcacg 1080

ttatggtggg cactctaagg agactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1140

caagtcatca tggcccttac ggcctgggct acacacgtgc tacaatggtc ggtacagagg 1200

gtcgccaagc cgcgaggtgg agctaatctc acaaaaccga tcgtagtccg gatcgcagtc 1260

tgcaactcga ctgcgtgaag tcggaatcgc tagtaatcgc gaatcagaat gtcgcggtga 1320

atacgttccc gggccttgta cacaccgccc gtcacaccat gggagtgggt tgcaccagaa 1380

gtagctagtc taaccttcgg gaggacggta ccaccggttg a 1421

Claims

1. Pseudomonas putida, which is classified and named as Pseudomonas putida (Pseudomonas putida) S9, has been preserved in China center for type culture Collection with the preservation number of CCTCC No. M2019635; the preservation date is as follows: 8, 8 and 16 in 2019; and (4) storage address: wuhan university in Wuhan, China.

2. The use of Pseudomonas putida S9 according to claim 1 for promoting the growth of Coprinus comatus.

3. Use according to claim 2, characterized in that it comprises the following steps: bacterial liquid culture, soil covering cultivation and fruiting management.

4. The use of claim 2, wherein the pseudomonas putida S9 is sprayed as a fermentation broth before the cultivation in the soil cover.

5. The use according to claim 4, wherein the concentration of Pseudomonas putida S9 in the fermentation broth is 1.5 x 10⁹cfu/mL～2.0×10⁹cfu/mL。

6. The use of claim 4, wherein the spraying amount of the fermentation liquor of the pseudomonas putida S9 is 500mL/m²。