CN109504611B - Bletilla striata endophytic fungus 1-G1 and application thereof - Google Patents

Abstract

The invention belongs to the technical field of microorganism application, and in particular relates to a strain of endophytic fungi 1-G1 and application thereof. A strain of endophytic fungus 1-G1, named Diaporthe spectabilis, was deposited in the Guangdong Provincial Microbial Culture Collection Center on June 6, 2018, and the deposit number is GDMCC NO.60382. In the present invention, an endophytic fungus (Diaporthe spectabilis) 1-G1 was isolated and screened from the medicinal plant B. radix for the first time. come with broad application prospects.

Description

An endophytic fungus 1-G1 and its application

technical field

The invention belongs to the technical field of microorganism application, and in particular relates to a strain of endophytic fungi 1-G1 and application thereof.

Background technique

The prevention and control of plant diseases in modern agriculture relies too much on the use of chemical pesticides. The application of a large number of chemical pesticides not only has a long-term destructive effect on the ecological environment, but also causes the quality of agricultural products to decline, pesticide residues exceeding standards, pathogenic bacteria resistance, and harmful to humans and animals, etc. many questions. It is of great significance to find safer and more effective disease control methods. The use of biological methods to control plant diseases can effectively solve the above problems.

Baiji Bletilla striata (Thunb.) Reichb.F. is a perennial herb of Orchidaceae Bletilla, named Baijier, Yangjiaoqi, Junqiuzi, Disuo, etc. Baiji is one of the traditional Chinese medicinal materials in my country. Hypertrophic and sticky tubers can be used as medicine, which has the functions of hemostasis, protection of mucous membranes, anti-tumor, antibacterial, etc. Few plant polysaccharides have astringent effects, so they are known as "must be astringent and harvested, enter the lungs to stop bleeding, and produce muscle to treat sores." It is the main ingredient of Chinese patent medicines such as Baiji syrup, Baiji granules, Weikangling and Kuaiwei tablets. In recent years, due to the over-exploitation and destruction of natural habitats of wild rhizomes, the resources of wild rhizomes have plummeted, and the yield and quality of rhododendrons cannot be guaranteed. It has been listed as one of the key protected wild medicinal plants by the state. One, it is now endangered. Due to the lack of endosperm in B. chinensis seeds, it is difficult to direct seedlings, and the survival rate of tissue culture seedlings is low. This makes it difficult to carry out white and large-scale planting.

At present, in the artificial cultivation process of white chia in Guangxi, wild white chia is mostly planted in tubers, and the large seedlings are planted for three years and harvested. However, the lack of quality standards for seedlings and the lag in research and development and application of varieties and quality control technologies are the key problems restricting the sustainable development of the Baiji industry. Therefore, it is of great significance to screen out the endophytic fungi that can promote the growth of A. chinensis. Screening and then developing and utilizing the endophytic fungi is also one of the measures to effectively ensure the quality of A. chinensis medicinal materials.

The information disclosed in this Background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an endophytic fungus 1-G1, which has the effect of promoting the growth of P. chinensis, thereby effectively promoting the growth of P. chinensis, increasing the yield, and further improving the quality of P. chinensis.

The technical scheme provided by the present invention is as follows:

An endophytic fungus 1-G1, named Diaporthe spectabilis, was deposited in the Guangdong Provincial Microbial Culture Collection Center on June 06, 2018, and the deposit number is GDMCC NO.60382.

The present invention also provides the application of the endophyte fungus 1-G1 of A. alba in promoting the growth of A. alba.

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

In the present invention, an endophytic fungus (Diaporthespectabilis) 1-G1 is isolated and screened from the medicinal plant Rhizoma Rhizoma for the first time. Broad application prospects.

Description of drawings

Fig. 1 is the morphological diagram of the colony of endophyte fungus 1-G1 of the present invention;

Fig. 2 is the determination of the content of IAA in the fermentation broth of endophyte fungus 1-G1 of the present invention;

Fig. 3 is the detection of siderophore synthesis ability of endophyte fungus 1-G1 active strain of the present invention;

Figure 4 shows the phylogenetic relationship of 24 strains of endophyte fungi and their similar fungi.

Description of preservation information

Diaporthe spectabilis, its preservation number is GDMCC NO.60382, the preservation date is June 6, 2018, and the preservation unit is the Guangdong Provincial Microbial Culture Collection Center (referred to as GDMCC, address: Laboratory Building, Provincial Institute of Microbiology, No. 100, Xianlie Middle Road, Guangzhou, China Fifth Floor, Postal Code: 510070).

Detailed ways

The following examples are for better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

The quantitative experiments in the following examples are all set to repeat the experiments three times, and the results are averaged.

Example 1 : Isolation, screening and identification of strains

1.1 Isolation of strains

Test materials: wild rhizomes collected from Guangxi Resource County and Huanjiang Autonomous County.

Medium: 1000ml potato dextrose agar medium (PDA medium); PDA medium contains potato 300g, glucose 20g, agar 20g, medium pH value is 6.0±0.2; DE medium contains 0.111mg CaCl ₂ , 0.174mg K _{2 SO4} , 27.8 mg _FeSO4.7H20 , 6.501 mg _MnCl2.4H20 , 0.241 _{mg NaMo4} , 0.123 mg MgSO4.7H20, _{0.0544 mg KH2PO4} , _{1.545 mg H3BO4} , _0.80516 mg ZnSO ₄ ·7H ₂ 0, 52.108 mg Na ₂ EDTA · 2H ₂ 0, 9 g soluble starch, 1 g yeast extract, 8 g agar, 1000 mL water, pH 6.0.

Surface disinfection: Gently shake off the soil and attachments on the roots and tubers of wild P. chinensis plants, rinse under running water, and place the washed roots and tubers on filter paper to absorb water for later use. On the ultra-clean workbench, put the roots and tubers of P. chinensis in sterile water, fully shake, wash and remove, and carry out surface disinfection in sequence. Rinse 4-5 times with sterile water. Using the conventional tissue block separation method, the roots and tubers of the sterilized surface of the tissue were placed on sterile filter paper, and the water was absorbed.

Isolation and purification of strains: Cut the sterilized roots and tubers into small pieces of 1 cm × 1 cm, inoculate the roots and tuber tissue blocks on the PDA plate medium respectively, and inoculate 4 tissue blocks in each petri dish. In a mold incubator, 28 ℃ constant temperature, dark, inverted culture. To ensure complete sterilization of the roots and tubers, use a sterile pipette to take the sterile water for the last cleaning of the roots and tubers and spread them on a new PDA medium, repeat 3 groups of petri dishes, and place them on the In a mold incubator, invert at 28°C for 7 days. If no bacteria grow on the PDA plate, it proves that the surface of the tissue is thoroughly disinfected, and the isolated fungi are endophytic fungi. After the tuber tissue was cultured on PDA medium for 2-3 days, hyphae could be observed to grow from the edge of the tissue block. When the hyphae grew to colonies visible to the naked eye, use a sterile toothpick to pick a small amount from the edge of the colony. The hyphae were inoculated on new PDA medium, and the transfer was repeated to obtain pure colonies.

The isolated endophytic fungi were inoculated into PDA plates and cultured at 28°C for 7-10 days before use.

1.2 Screening

The symbiotic culture of aseptic seedlings and endophytic fungi was used to screen the tested endophytic fungi for promoting activity.

Prepare the symbiotic medium in advance according to the DE medium formula, divide it into 250mL tissue culture bottles, and sterilize each bottle at 121°C for 30min for later use. On the ultra-clean workbench, take out the sterile tissue culture seedlings of P. chinensis, wash the medium carried out by the roots with sterile water, place them on sterile filter paper to absorb the water, and then transfer them into DE medium. Each bottle receives 4 strains, arranged in the center of the word "mouth". After the transfer is completed, it is placed in the culture room for 15 days, the temperature is 25-26 ℃, the light is 8-12 hours a day, and the light intensity is 2000-3000Lux. After the sterile seedlings were grown on DE medium for 15 days, their growth status was basically stable. At this time, a fungus cake with a diameter of 0.6 cm was taken from the edge of the activated endophytic fungus mother colony as the inoculum material, and the fungus cake was picked and inoculated into the middle of 4 Radix Radix et Rhizoma seedlings, about 2 cm away from the seedlings. One piece of tissue culture bottle was inoculated, and each strain was repeated for 3 bottles, and the control group was inserted into a sterile PDA agar block. After the inoculation is completed, it is sealed and placed in a culture room for cultivation at a temperature of 25-26°C, 8-12 hours of light per day, and the light intensity is 2000-3000Lux. Regularly observe the growth status of fungi and P. chinensis tissue culture seedlings. After 30 days of symbiotic culture, the fungi that can grow well with the sterile seedlings of Rhizoma Rhizoma were selected based on the moderate growth rate of bacteria and the normal growth of sterile seedlings of Rhizoma Rhizoma. The strains obtained from the primary screening were re-inoculated into DE medium in the same way, with 3 sterile seedlings per bottle, and sterile agar blocks of the same size were inserted as controls. Under the same culture conditions, after symbiotic culture for 60 days, the control seedlings and symbiotic seedlings were respectively taken out, the residual medium at the roots was washed, and the surface water was blotted with filter paper. The plant height, the number of new roots, the number of new shoots, and the diameter of tubers were measured.

Results A total of 5 growth-promoting fungal strains were obtained, which had strong growth-promoting effect on Baiji, and one of them was named 1-G1.

1.3 Identification

(1) Morphological characteristics of strains

Observation of morphological characteristics of bacteria: The endophyte fungal strain 1-G1 to be identified was inoculated on PDA medium, cultured at 28°C, and its culture characteristics and color changes were observed at 5, 14 and 20 days, respectively. Take stable and mature color characteristics as its culture characteristics, as the basis for identification. Observe and record the color of aerial hyphae, colony size, color, tissue shape, surface shape, etc. as reference features.

As shown in Figure 1, the morphological characteristics of the colony: on the PDA medium, the colony is round, the hyphae in the medium are gray-black and radial, and the aerial hyphae are gray-black and cotton-wool.

(2) Strain ITS sequence and its phylogenetic analysis

Preparation of DNA template:

Reagents: (1) Lysis buffer: Tris-Ac (pH 8.0) 20mM, 3% CTAB, 1.4mol/L NaCl, 10% DTT); (2) saturated phenol:chloroform:isoamyl alcohol (25:24:1) ) mixed solution, isopropanol, ddH O;

DNA extraction:

Take an appropriate amount of fungal hyphae, add 600 μL CTAB extraction buffer (20 mmol/L Tris (pH=8.0), 3% CTAB, 1.4 mol/L NaCl, 10% DTT), add a little sterilized quartz sand, and fully grind the hyphae . Transfer the grinding liquid into a 1.5 mL EP tube, and place it in a water bath at 65°C for 30 min, during which the EP tube is shaken every 10 min to mix the grinding liquid. Water bath at 65°C and ice bath for 10 min, add 400 μL of saturated phenol:chloroform:isoamyl alcohol (25:24:1) mixture, and mix well. Centrifuge at 12000rpm for 10min. Take 400 μL of supernatant, add pre-cooled isopropanol, mix well, let stand, ice bath for 1 hour, centrifuge at 12,000 rpm for 10 min, discard the supernatant, air dry, resuspend with 20 μL of ddH2O, and then take 2 μL as PCR template ITS sequences were amplified by PCR.

(1) PCR instrument: ABI 2720-XL DNA sequencer (Applied Biosystems, USA)

(2) Amplification primers: ITS1 (5′-T C C G T A G G T G A A C C T G C G G-3′) and ITS4 (5′-T C C T C C G C T T A T T G A T A T G C-3′)

(3) Amplification system: The ITS sequence PCR amplification system is shown in Table 1.

Table 1 ITS sequence PCR amplification system

Reactant Injection volume 2X TagMasterMix 25μL Primer-1 1μL Primer-2 1μL Template DNA 2μL ddH₂O 21μL total reaction volume 50μL

PCR reaction conditions are shown in Table 2.

Table 2 PCR reaction conditions

NOTE: Step 2 is performed for 30 cycles.

Electrophoresis detection of PCR amplification products:

The electrophoresis conditions are 1% agarose gel (containing 1 μL/10 mL of Super GelRed 10000× aqueous solution), 1× TBE electrophoresis buffer, electrophoresis at 90V for 1 hour, and the loading volume of PCR product is 4 μL, which is mixed with 1 μL Loading dye Sample later.

The results were observed under the UV lamp of the gel imaging system, and the DL1000DNA Marker of TaKaRa Company was used as the reference for nucleic acid standard molecular weight to determine the length of the amplified fragment. The amplified product band should be at the 400-700bp position of the standard.

PCR product purification and sequencing: carried out by Shenzhen Huada Gene Technology Co., Ltd.

Construction of phylogenetic tree:

Log in to NCBI, compare the fungal rDNA ITS sequence obtained by sequencing with the sequence in the GenBank database, record its accession number, the most similar strain, similarity, etc., and download the rDNA ITS sequence of the strain, and use MEGA 7 for cluster analysis and construct the NJ phylogenetic tree. The ITS sequence of 1-G1 is shown in SEQ ID No.1.

Blast homology alignment of the rDNA ITS sequence in GeneBank to obtain the representative strain with the highest similarity, record its accession number, most similar strain, similarity, etc., and download the rDNA ITS sequence of the strain to construct the NJ phylogeny tree (shown in Figure 4). The most similar strains to strain 1-G1 are shown in Table 3.

Table 3. Strains with the closest similarity to fungus 1-G1

Using primers ITS1 and ITS4, a fragment of 500-600 bp in size was amplified from the genomic DNA of the strain, which was sequenced and the sequencing results were compared by BLASTn in GenBank.

The results show that the strain 1-G1 has a high base sequence similarity with the fungi in the genus Diaporthe. Therefore, the reference strain sequence of the genus Diaporthe in GenBank was downloaded for phylogenetic analysis. Diaporthe sp. (KX110392 ) as an outgroup. In the constructed phylogenetic tree, 1-G1 clustered with multiple lines of Diaporthe sp. to form a terminal branch with a support strength of 99%.

The comparison results of base similarity showed that there was no difference in base sequence between 1-G1 and Diaporthe sp., and the sequence similarity was 100%. Based on morphological and molecular biological characteristics, the strain was preliminarily identified as Diaporthe sp..

Example 2 : Detection of growth-promoting properties of active strains

2.1 Detection of indoleacetic acid (IAA) synthesis ability of active strain 1-G1 fermentation broth

Accurately weigh 1 mg of IAA, add 10 mL of distilled water to dissolve, and obtain a 100 μg/mL IAA reference solution. The serial dilution of IAA reference solution is 10, 20, 30, 40, 50, 60, 70, 80ug/mL. The IAA solution of each gradient and the Salkowski colorimetric solution were mixed at a volume ratio of 1:1, and the OD530 of each concentration of IAA solution was measured after being placed in the dark at room temperature for 30 min. Taking the concentration of IAA as the abscissa and the OD530 of each concentration of IAA solution as the ordinate, draw a standard curve.

Add 20 mL of IAA detection medium to a 50 mL sterilizable centrifuge tube, sterilize at 121 °C for 20 min, and take it out for cooling. On the ultra-clean workbench, use a hole punch to punch out 0.6 cm of bacterial blocks at the edge of the activated parent colony, and use sterile toothpicks to inoculate the bacterial blocks into the above centrifuge tubes, with two inoculated in each test tube. , repeat 3 tubes, and inoculate sterile agar blocks of the same size as blank controls. Incubate at 28°C on a shaker at 150 r/min for 5 days. Take 100 μL of culture droplets onto a white ceramic plate, add 100 μL of Salkowski colorimetric solution, and place the white ceramic plate at room temperature for 30 minutes in the dark to observe the color change. For the control, only add 100 μL of sterile medium to the colorimetric solution. The color solution was added with 50mg/L and 25mg/L of IAA. The strain culture solution with obvious color change was centrifuged at 8000 r/min for 10 min, and 2 mL of the supernatant was passed through a 0.22 μm microporous membrane. After filtration and sterilization, 2 mL of Salkowski colorimetric solution was added. The blank IAA detection medium was treated with the same treatment as the control and zeroed, and the OD530 of each mixture was measured, and the IAA content of the sample was calculated with reference to the IAA standard curve obtained above, with 3 replicates, and the unit of IAA content was μg/mL.

As shown in Figure 2, in the positive control from the left, 100 μg/mL and 25 μg/mL of IAA solution were added to 100 μL Salkowski colorimetric solution, and after shading for 30 min, the color of the mixture changed from colorless to pink, and the higher the concentration of IAA, The higher the color, the darker the color. The fungal 1-N2 fermentation broth was centrifuged, and the supernatant was mixed with the Salkowski colorimetric solution. After shading for 30 minutes, the mixture had an obvious pink change.

The fermentation broth of fungus 1-G1 was used for IAA content determination, and the result showed that the IAA content was 42.38 μg/mL. The growth-promoting effect of IAA is mainly to promote the growth of cells, especially the elongation of cells, which can promote the rooting of cuttings. In the symbiosis and pot experiments, the fungus 1-G1 showed obvious promoting activity. In this test, the strain 1-G1 has a strong ability to synthesize IAA, which is consistent with the results of the previous fungi growth promotion test. .

2.2 Detection of siderophore synthesis ability of strains

Add 20 mL of iron-free Charlie's medium to a 50 mL sterilizable centrifuge tube, sterilize at 121°C for 20 min, and take it out for cooling. On the ultra-clean workbench, use a hole punch to punch a 0.6cm block of bacteria from the edge of the activated parent colony, and inoculate the bacteria block into the above centrifuge tubes respectively. A sterile agar block of the same size was added to the control, and cultured at 28°C at 150 r/min for 5 days. Centrifuge the culture solution at 8000rpm for 10min, take the supernatant, put the supernatant and CAS color developing solution on a white ceramic plate in a volume ratio of 1:1, shake and mix gently, observe the color change, and record the color change of the liquid after mixing. For red, purple or yellow culture medium, and take pictures to record. Centrifuge the culture liquid of the strain whose color changes to red, purple or yellow for 10min (8000rpm), take 2mL of the supernatant to pass through a 0.22μm microporous membrane, filter and sterilize, add 2mL of CAS colorimetric solution, and let stand at room temperature for 1h Then, use distilled water to zero, measure its OD680, count as "As", take 2 mL of sterile iron-free Chaplain culture medium, measure its OD680 as a reference value according to the same method, count as "Ar". The concentration of siderophore in the culture medium was determined by the change of the OD680 value of the blank control. The siderophore concentration in the test is expressed in siderophore activity units (SU), SU=[(Ar-As)/Ar]×100%. The larger the SU value, the stronger the ability to synthesize siderophore, the As/Ar represents the relative content of siderophore in the culture medium, and the smaller the value, the higher the siderophore content. Generally, the As/Ar of bacteria with high siderophore production capacity is less than 0.5.

In nature, iron mostly exists in the form of FeO and ferric hydroxide complexes, which are difficult for organisms to use in this insoluble state. Siderophores are a class of small-molecule metal ion chelates produced by microorganisms, which have a strong affinity for Fe3 ⁺ .

As shown in Figure 3, among the 5 endophytic fungi, the fungus 1-G1 bacterial suspension was mixed with CAS chromogenic solution to give a red reaction, and after the sterile culture medium was mixed with CAS, the color was light green. It shows that the fungus 1-G1 has the ability to synthesize siderophore.

Table 4 Quantitative determination of siderophore of strains

As shown in Table 4, the SU of strain 1-G1 was 0.5539, and the As/Ar value was less than 0.5, indicating that the strain was a high siderophore-producing strain.

In conclusion, the strain of endophyte 1-G1 has strong ability to produce IAA, and has the ability to synthesize siderophore. Moreover, it played a significant role in promoting growth in the seedling growth promotion test. Therefore, in terms of ecological planting of B. serrata, the endophytic fungus 1-G1 fermentation broth of B. serrata has obvious potential.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise form disclosed, and obviously many changes and modifications are possible in light of the above teachings. The exemplary embodiments were chosen and described for the purpose of explaining certain principles of the invention and their practical applications, to thereby enable one skilled in the art to make and utilize various exemplary embodiments and various different aspects of the invention. Choose and change. The scope of the invention is intended to be defined by the claims and their equivalents.

sequence listing

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Claims (2)

1. A bletilla striata endophytic fungus 1-G1, which is characterized in that the bletilla striata endophytic fungus is classified and named as a diapositive shell (A)Diaporthe sp.) And is preserved in Guangdong province microbial culture collection center in 2018, 06 months, with the preservation number being GDMCC NO. 60382.

2. The use of bletilla striata endophytic fungus 1-G1 in promoting growth of bletilla striata according to claim 1.

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