Bacillus subterraneus X18 and application thereof in promoting sclerotium formation of morchella esculenta

Abstract

The invention discloses bacillus subterraneus X18 and application thereof in promoting sclerotium formation of morchella esculenta. The subterranean bacillus X18 can promote sclerotium formation in a morchella sextelata strain, the morchella sextelata and the strain X18 are co-cultured on a PDA plate, the sclerotium of the morchella sextelata becomes larger, the dry weight is increased by 86.3%, and the dry weight of the sclerotium is increased by 25.6%-33.4% when the strain X18 is added into a morchella sextelata stock. Meanwhile, the invention also optimizes the fermentation process of the strain, has simple conditions, is convenient to apply, and is suitable for large-scale production of the morchella strain.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a subterranean bacillus X18 that promotes sclerotium formation of Morchella sutchuenensis and an application method thereof. Background Art

[0002] Morchella spp. belongs to the phylum Ascomycota, class Peziomycetes, orders Peziales, family Morchaceae, genus Morchella in taxonomy. It is named because of its concave appearance that resembles a sheep's tripe. Morchella has delicious and tender flesh, and has high nutritional and medicinal value. It is a well-known food supplement. Liumei Morchella is a black Morchella variety that has been successfully domesticated in China's field cultivation model in recent years and has been cultivated on a large scale commercially.

[0003] Sclerotia is a dormant body structure formed by closely connected and interwoven hyphae. The sclerotia of Morchella is an important specific form of Morchella hyphae. The main function of sclerotia is to resist high temperature, low temperature, dryness and other environments that are not conducive to the growth of Morchella. Under suitable conditions, it can germinate and produce fruiting bodies or mycelium. In 1982, Ower first realized the artificial cultivation of Morchella by cultivating Morchella sclerotia and then inducing sclerotia to germinate and produce ascocarps. Sclerotia is a necessary stage in the life history of Morchella, and inducing the formation of sclerotia is the basis of artificial cultivation of Morchella. Different Morchella species, different strains of the same Morchella species, and the same Morchella strain under different conditions have different abilities to form sclerotia. At present, the mechanism of sclerotia formation and expression regulation of Morchella still needs to be systematically studied. Summary of the invention

[0004] The primary purpose of the present invention is to provide a strain of Bacillus subterraneus X18 which can promote the formation of sclerotia in Morchella species, and the deposit number is: CGMCC No.30745.

[0005] The second object of the present invention is to provide the use of the subterranean Bacillus X18 in promoting sclerotia formation in Morchella species.

[0006] Furthermore, the morel is Morchella oleracea.

[0007] The formula of the Morchella culture medium is: 62% wheat, 18% sawdust, 10% humus, 8% wheat bran, 1% quicklime and 1% gypsum. The six-sister Morchella cake is inoculated on the original culture medium, and X18 bacterial liquid is inoculated at the same time, and cultured at a constant temperature of 18-22°C in the dark for at least 30 days.

[0008] Furthermore, the culture medium formula is: 62% wheat, 18% sawdust, 10% humus, 8% wheat bran, 1% quicklime, and 1% gypsum. The volume of the glass bottle is 500 ml, and the culture medium is 600 g. Four Morchella lilii with a diameter of 10 mm are inoculated on the original culture medium, and 4-8 ml of X18 culture solution cultured overnight are inoculated at the same time. The OD 600 ≥2.0, culture at 20℃ in the dark for 30 days.

[0009] The third object of the present invention is to provide a fermentation and cultivation method of the subterranean Bacillus X18, wherein the fermentation medium formula is: beef extract 5.0 g / L, peptone 10 g / L, bean cake powder 2.0 g / L, glucose 5.0 g / L, sodium chloride 5.0 g / L, potassium dihydrogen phosphate 0.10 g / L, and the fermentation conditions are: inoculation amount 2.5-3.5%, temperature 28-32°C, liquid volume 60-80%, initial pH 6.5-7.5, rotation speed 220-250 rpm / min, and cultivation under these conditions is 16-24 h.

[0010] Furthermore, the fermentation medium formula was: beef extract 5.0 g / L, peptone 10 g / L, soybean cake powder 2.0 g / L, glucose 5.0 g / L, sodium chloride 5.0 g / L, potassium dihydrogen phosphate 0.10 g / L, and the fermentation conditions were: inoculation amount 3%, temperature 30°C, liquid volume 70%, initial pH 7.5, rotation speed 250 rpm / min, and culture was carried out under these conditions for 24 h.

[0011] The underground Bacillus X18 of the present invention has a deposit number of CGMCC No. 30745. The strain can significantly increase the sclerotia yield in the six-sister morel species. The strain X18 is isolated and screened from the soil of the six-sister morel fungus base. The six-sister morel and the strain X18 are co-cultured on a PDA plate, and the sclerotia yield of the morel is increased by 86.3%. The strain X18 is added to the original six-sister morel species, and the sclerotia yield is increased by 33.4%. The strain X18 was preliminarily identified as Bacillus subterraneus by 16S rDNA sequence analysis. The fermentation medium formula of strain X18 was optimized as follows: beef extract 5.0 g / L, peptone 10 g / L, soybean meal powder 2.0 g / L, glucose 5.0 g / L, sodium chloride 5.0 g / L, potassium dihydrogen phosphate 0.10 g / L. The optimal fermentation conditions were inoculation volume 3%, temperature 30℃, liquid volume 70%, initial pH 7.5, rotation speed 250 rpm / min. Under these conditions, the number of effective viable bacteria reached 34.4×10-10 after 24 h of culture. 9 cfu / mL, which was increased by 98.8% compared with the initial fermentation medium, and the optimization efficiency was significant.

[0012] In conclusion, the addition of Bacillus subterraneus X18 in the present invention can significantly increase the sclerotia yield in the Morchella liliifolia strain. The fermentation conditions of this strain are simple, the application is convenient, and it is suitable for the large-scale production of Morchella liliifolia strains.

[0013] The deposit information of the strain of the present invention is as follows: it was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration Committee on May 23, 2024, with the deposit number: CGMCC NO.30745, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. Classification name: Bacillus subterraneus X18.

[0014] Advantages of the present invention:

[0015] 1. The strain X18 described in the present invention is the first reported growth-promoting microorganism that can significantly increase the sclerotia yield in Morchella spp., which is beneficial to improving the quality of Morchella spp.

[0016] 2. The present invention optimizes the method for expanding and culturing strain X18, which has simple fermentation conditions, strong reproduction ability and low fermentation cost; the fermentation liquid can be used directly, is harmless to humans and animals, does not pollute the environment, and does not require post-treatment, and has broad application prospects for industrial production.

[0017] 3. Currently, the function of underground Bacillus is mainly used for biological control. This invention is the first report that underground Bacillus can be used in the field of edible fungi. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :The effects of three strains on the growth promotion of Morchella liumei on PDA plates (A) and the dry weight of sclerotia (B);

[0019] Figure 2 : Colony morphology (A), bacterial microscopic image (B) and phylogenetic tree (C) of strain X18;

[0020] Figure 3 :Effect of strain X18 on the growth promotion of Morchella truncatula on PDA plates

[0021] Figure 4 :The effect of strain X18 on the sclerotium growth promotion of Morchella liumei original species;

[0022] Figure 5 : Optimization of carbon source types (A), nitrogen source types (B), and inorganic salt types (C) in the liquid fermentation medium of strain X18 and the growth curve under optimized conditions (D). DETAILED DESCRIPTION

[0023] The present invention will be further described below with reference to the embodiments, but will not be limited to the present invention.

[0024] Example 1 Screening and identification of the growth-promoting sclerotium strain X18 of Morchella liumei

[0025] 1. The bacteria were isolated and purified using beef extract peptone medium with the following formula: beef extract 3.0 g / L, peptone 10.0 g / L, sodium chloride 5.0 g / L, agar 20 g / L, pH 7.5.

[0026] 2. Collect the soil from the base of the morel when it is fruiting. Take 2 cm from the bottom of each fruiting body. 3 For soil, 10 soil samples were mixed and crushed, then passed through a 100-mesh sieve. 10 g of soil sample was added to 90 ml of sterile water containing glass beads, and shaken at 100 rpm for 30 min. After the sample was dispersed, it was allowed to stand for 5 min. 0.5 ml of soil suspension was taken and added to 4.5 ml of sterile water and shaken to obtain 10 -2 Dilution suspension, dilute to 10 by 10-fold dilution method -6 , take 10 -4 , 10 -5 , 10 -6 Three gradients.

[0027] 3. Pipette 0.1 ml onto the separation and purification medium plate, culture at 30℃ for 3-4 days, pick single colonies with different morphologies on the separation and purification plate, streak and preserve them, and obtain 182 strains after preliminary separation and purification.

[0028] 4. 182 strains were cultured in beef extract peptone medium without agar for 1-2 days. 600 When the value was ≥2.0, 50 μl of bacterial solution (the control CK was 50 μl of sterilized beef extract peptone medium) and the six-sister Morchella strain were inoculated into PDA medium plates at the same time. The mycelial growth of Morchella was observed for 2-3 days. After 10 days of cultivation, Morchella sclerotia were picked out from each plate for comparison. The results showed that the sclerotia diameter of Morchella with strain X18 was larger than that of the control ( Figure 1 A).

[0029] 5. Identification of X18 strain: Strain X18 forms moist, raised colonies on beef extract peptone medium. The colonies are light yellow with irregular and translucent edges and a colony diameter of 1-1.5 mm ( Figure 2 A); Microphotographs show that strain X18 is short rod-shaped or oval, spore-forming, and Gram-positive ( Figure 2B); extract the genomic DNA of strain X18, use the bacterial 16S rDNA universal primer 27F / 1492R for PCR amplification, and compare the sequencing results with the GeneBank database by Blast. The results show that the sequence homology of strain X18 of the present invention and Bacillus subterraneus strain: A6, GenBank: KY202699.1 16S rDNA is 99.79%. Nine representative strains of the genus Bacillus were selected together with X18 to construct a phylogenetic evolutionary tree ( Figure 2 C), the results showed that X18 had the closest evolutionary relationship with Bacillus subterraneus, and strain X18 was preliminarily identified as Bacillus subterraneus.

[0030] Bacillus subterranean X18, Pseudomonas putida X14 (a growth-promoting microorganism that can increase the growth rate of Morchella mycelium, patent number: ZL202010461696.3), Bacillus subterranean A6 and Morchella lilii strain were inoculated onto PDA culture plates at the same time. After 10 days of culture, Morchella sclerotia were picked out from each plate, washed with sterile water and dried. The morphology and dry weight of sclerotia were compared. The results showed that the Morchella sclerotia with the addition of strain X18 not only had a larger diameter ( Figure 1 A), and the total dry weight of sclerotia in 10 plates (0.680±0.072 g) increased significantly by 86.3% compared with the control (0.365±0.095 g) (P<0.01). There was no significant difference in the morphology of sclerotia between strains X14 and A6, and the total dry weight (X14=0.410±0.159 g, A6=0.396±0.072 g) was also not significantly different from the control ( Figure 1 B). Strains X14 and A6 were purchased from the Hunan Agricultural Microbial Germplasm Resource Bank.

[0031] Bacillus subterraneus X18 and Morchella tiliacea strain were inoculated on PDA medium plates at the same time. After 5 days of cultivation, a large number of sclerotia appeared, which were mainly concentrated around the inoculation block in a radial pattern. After 10 days of cultivation, the sclerotia of Morchella tiliacea were picked out from the plate, cleaned with sterile water and dried. The morphology and dry weight of the sclerotia were compared. The results showed that there was no significant difference in the size and dry weight of the sclerotia of Morchella tiliacea with strain X18 (1.492±0.291 g) and the control (1.385±0.267 g) ( Figure 3 ).

[0032] Example 2 Determination of the Growth-Promoting Effect of Bacillus subterraneus X18 on Sclerotia in Morchella liumei Original Species

[0033] Wheat grains were used to prepare the original culture medium of Morchella, and the culture medium formula was: 62% wheat, 18% sawdust, 10% humus, 8% wheat bran, 1% quicklime, and 1% gypsum. The volume of the glass bottle was 500 ml, and the culture medium was 600 g. Four Morchella lilii mushroom cakes with a diameter of 10 mm were inoculated on the original culture medium with a hole puncher, and 1 ml, 2 ml, 4 ml, and 8 ml of X18 bacterial solution cultured overnight by shaking were inoculated respectively. The OD 600 ≥2.0 (CK was an equal volume of sterilized beef extract peptone medium), and the sclerotial yield of Morchella was compared after culturing at 20℃ in the dark for 30 days ( Figure 4 The results showed that the sclerotium yield in the original culture with 4 ml of X18 bacterial solution added (113.0±17.1 g) increased by 33.4% (P<0.05) compared with the control (84.7±18.0 g), and the sclerotium yield in the original culture with 8 ml of X18 bacterial solution added (114±11.4 g) increased by 25.6% (P<0.05) compared with the control (90.8±18.6 g).

[0034] Example 3 Optimization of fermentation culture process of Bacillus subterraneus X18

[0035] The preserved Bacillus subterraneus X18 was streaked onto beef extract peptone medium and cultured at 30°C for 24 h. A single colony was picked and inoculated into beef extract peptone medium without agar and cultured overnight at 30°C and 250 rpm / min for later use as seed solution.

[0036] Initial fermentation medium: beef extract 3.0g / L, peptone 10.0g / L, sodium chloride 5.0g / L, pH=7.5. 0.5% glucose, maltose, sucrose, and soluble starch were used as carbon source medium for fermentation of strain X18. The results showed that the number of effective live bacteria was the highest when soluble starch was used as carbon source, reaching 25.6×10 9 cfu / ml; followed by glucose, which is 24.4×10 9 cfu / ml; when sucrose and maltose were used as carbon sources, the value was significantly reduced to 17.2×10 9 cfu / ml, 12.4×10 9 cfu / ml( Figure 5 A). Considering that the cost of glucose raw material is lower than that of soluble starch, and there is no significant difference in the number of effective viable bacteria between the two, glucose was selected as the best carbon source.

[0037] The fermentation culture of strain X18 was compared using 2% peptone, beef extract, yeast extract, and soybean cake powder as nitrogen source culture media. The results showed that the number of effective live bacteria was the highest when soybean cake powder was used as the nitrogen source, reaching 21.2×10 9cfu / ml; when the other three were used as nitrogen sources, the effective viable bacteria count was significantly reduced to 10.8×10 9 cfu / ml~13.3×10 9 cfu / ml( Figure 5 B). Therefore, soybean cake powder was selected as the best nitrogen source.

[0038] The fermentation culture of strain X18 was compared using 0.1% dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate as the inorganic salt components of the culture medium. The results showed that the number of effective live bacteria was the highest when potassium dihydrogen phosphate was used as the inorganic salt for fermentation, which was 19.6×10 9 cfu / ml; followed by potassium dihydrogen phosphate, which is 18.5×10 9 cfu / ml; when the other two were used as inorganic salts, the effective viable count was significantly reduced to 14.0×10 9 cfu / ml~14.2×10 9 cfu / ml( Figure 5 C). Therefore, potassium dihydrogen phosphate was selected as the best inorganic salt. The optimized culture medium formula was obtained: beef extract 5.0 g / L, peptone 10 g / L, soybean cake powder 2.0 g / L, glucose 5.0 g / L, sodium chloride 5.0 g / L, potassium dihydrogen phosphate 0.10 g / L.

[0039] The fermentation conditions were further optimized through experiments. The inoculation amount was 3%, the temperature was 30℃, the liquid volume was 70%, the initial pH was 7.5, and the rotation speed was 250 rpm / min. The strain was in the logarithmic growth phase, and the number of viable bacteria increased rapidly. Then it entered the stable phase and reached the optimal growth period after 24 hours of cultivation. The effective viable bacteria count was 34.4×10 9 cfu / mL( Figure 5 D), compared with the initial fermentation medium (17.3×10 9 cfu / mL) increased by 98.8%, and the optimization efficiency was significant.

Claims

1. A strain of Bacillus subterraneus X18, with the deposit number: CGMCC No.30745.

2. Use of the Bacillus subterraneus X18 described in claim 1 in promoting sclerotia formation in Morchella species.

3. The use according to claim 2, characterized in that: The morel is Morchella esculenta.

4. The use according to claim 2 or 3, characterized in that: The culture medium formula is: 62% wheat, 18% sawdust, 10% humus, 8% wheat bran, 1% quicklime, and 1% gypsum. The morel cake is inoculated on the original culture medium, and the X18 bacterial solution is inoculated at the same time. The culture is kept at a constant temperature of 18-22℃ and protected from light for at least 30 days.

5. The use according to claim 4, characterized in that The culture medium formula is: 62% wheat, 18% sawdust, 10% humus, 8% wheat bran, 1% quicklime, and 1% gypsum. The glass bottle volume is 500 ml, and the culture medium is 600 g. Four 10mm diameter Morchella lilii cakes are inoculated on the original culture medium, and 4-8 ml of overnight cultured X18 culture liquid is inoculated at the same time. The OD 600 ≥2.0, culture at 20℃ in the dark for 30 days.

6. The fermentation culture method of Bacillus subterraneus X18 according to claim 1, characterized in that: Fermentation medium formula: beef extract 5.0 g / L, peptone 10 g / L, soybean cake powder 2.0 g / L, glucose 5.0 g / , sodium chloride 5.0 g / L, potassium dihydrogen phosphate 0.10 g / L, fermentation conditions are inoculation amount 2.5-3.5%, temperature 28-32℃, liquid volume 60-80%, initial pH 6.5-7.5, rotation speed 220-250 rpm / min, and culture under this condition for 16-24 h.

7. The fermentation culture method according to claim 6, characterized in that: The fermentation medium formula was as follows: beef extract 5.0 g / L, peptone 10 g / L, soybean cake powder 2.0 g / L, glucose 5.0 g / L, sodium chloride 5.0 g / L, potassium dihydrogen phosphate 0.10 g / L. The fermentation conditions were inoculation volume 3%, temperature 30°C, liquid volume 70%, initial pH 7.5, rotation speed 250 rpm / min, and culture was carried out for 24 h under these conditions.

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