CN109504611B

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

Info

Publication number: CN109504611B
Application number: CN201811588958.1A
Authority: CN
Inventors: 李良波; 田佩雯; 黄荣韶
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2018-12-25
Filing date: 2018-12-25
Publication date: 2020-11-03
Anticipated expiration: 2038-12-25
Also published as: CN109504611A

Abstract

The invention belongs to the technical field of microorganism application, and particularly relates to a bletilla striata endophytic fungus 1-G1 and application thereof. A bletilla striata endophytic fungus 1-G1 is classified and named as Diaporthe spectabilis, and is deposited in Guangdong province microorganism culture collection center in 2018 in 06 months, with the deposition number being GDMCC NO. 60382. The invention separates and screens a strain of endophytic fungi (Diaporter spectabilis)1-G1 from the interior of the medicinal plant bletilla striata for the first time, the strain has stronger growth promotion effect on the growth of the bletilla striata and all aspects, and wide application prospect is brought for the yield and quality control of the bletilla striata.

Description

Bletilla striata endophytic fungus 1-G1 and application thereof

Technical Field

The invention belongs to the technical field of microorganism application, and particularly relates to a bletilla striata endophytic fungus 1-G1 and application thereof.

Background

The prevention and control of plant diseases in modern agriculture excessively depends on the use of chemical pesticides, and the application of a large amount of chemical pesticides not only has long-term destructive effect on the ecological environment, but also causes various problems of quality reduction of agricultural products, overproof pesticide residues, drug resistance of pathogenic bacteria, harm to people and livestock and the like. The method has great significance for finding safer and more effective disease control methods, and the problems can be effectively solved by using biological methods to control plant diseases.

Bletilla striata (Thunb.) Reichb.F. is a perennial herb of Bletilla striata of the family Orchidaceae, named white chicken, rhizoma homalomenae, fructus et radix Polygalae, radix seu folium Cayratiae Oligocarpae, rhizoma Bletilla is one of the traditional Chinese medicinal materials in China, and the tuber with fleshy and sticky meat can be used as a medicine, has the effects of stopping bleeding, protecting mucosa, resisting tumor, resisting bacteria and the like, and rarely has the effect of plant polysaccharide with convergence, so the rhizoma Bletilla has the name of' must astringe and accept, enter lung to stop bleeding, promote tissue regeneration and treat sore, and is the best surgical name, and is the main raw material of Chinese patent medicines such as rhizoma Bletilla syrup, rhizoma Bletilla granules, stomach-recovering tablet, and fast-stomach tablet and the like. In recent years, as wild bletilla striata is over-developed and destroyed by natural habitat, the wild bletilla striata resource is reduced linearly, the yield and the quality of bletilla striata medicinal materials can not be guaranteed, and the wild bletilla striata is one of wild medicinal plants which are listed as key protection by the state and is endangered at present. As the bletilla striata seeds have no endosperm, the direct seeding and seedling formation are difficult, and the survival rate of the tissue culture seedlings is low. All of which make it difficult to perform large-scale planting of bletilla striata.

At present, wild bletilla striata is planted in tuber branches in the artificial cultivation process of bletilla striata in Guangxi areas, and large seedlings are planted and harvested for three years. But the lack of seedling quality standards and the lag in research and development and application of varieties and quality control technologies are key problems for restricting the sustainable development of rhizoma bletillae industries. Therefore, screening the endophytic fungi capable of promoting the growth of the bletilla striata has great significance, and screening and further developing and utilizing the bletilla striata endophytic fungi is also one of measures for effectively ensuring the quality of the bletilla striata 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 skilled in the art.

Disclosure of Invention

The invention aims to provide a bletilla striata endophytic fungus 1-G1, which has the effect of promoting growth of bletilla striata, so that growth of bletilla striata can be effectively promoted, yield can be increased, and quality of bletilla striata can be further improved.

The technical scheme provided by the invention is as follows:

a bletilla striata endophytic fungus 1-G1 is classified and named as Diaporthe spectabilis, and is deposited in Guangdong province microorganism culture collection center in 2018 in 06 months, with the deposition number being GDMCC NO. 60382.

The invention also provides application of the bletilla striata endophytic fungus 1-G1 in promoting growth of bletilla striata.

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

the invention separates and screens a strain of endophytic fungi (Diaporter hepatilis) 1-G1 from the interior of medicinal plant bletilla striata for the first time, the strain has stronger growth promotion effect on white and all-side growth, and wide application prospect is brought for the yield and quality control of bletilla striata.

Drawings

FIG. 1 is a colony morphology of the bletilla striata endophytic fungus 1-G1 of the present invention;

FIG. 2 is a graph showing the content determination of IAA in the fermentation broth of the active strain of bletilla striata endophytic fungi 1-G1;

FIG. 3 is the test of the synthesizing ability of the siderophore of the active strain of bletilla striata endophytic fungus 1-G1;

FIG. 4 shows the phylogenetic relationship between 24 strains of bletilla striata and endophytic fungi and their analogous fungi.

Description of preservation information

Diaporthe spectabilis, the preservation number is GDMCC NO.60382, the preservation date is 06 months in 2018, and the preservation unit is Guangdong province microorganism culture preservation center (GDMCC for short, address: Wulou of Michelia Summinck & gt, Michelia Tokyo, 100 province microorganism institute, China, postal code: 510070).

Detailed Description

The following examples are given to better understand the present invention and are not intended to limit the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

In the quantitative experiments in the following examples, three replicates were set up and the results averaged.

Example 1: isolation, screening and identification of strains

1.1 isolation of the Strain

Test materials: wild bletilla striata collected from Guangxi resource county and Yunjiang autonomous county.

Culture medium: 1000ml of potato dextrose agar medium (PDA medium); the PDA culture medium contains 300g of potato, 20g of glucose and 20g of agar, and the pH value of the culture medium is 6.0 +/-0.2; DE Medium contained 0.111mg CaCl₂，0.174mg K₂SO₄，27.8mg FeSO₄·7H₂0，6.501mg MnCl₂·4H₂0，0.241mg NaMo₄，0.123mg MgSO₄·7H₂0，0.0544mg KH₂PO₄，1.545mg H₃BO₄，0.80516mg ZnSO₄·7H₂0，52.108mg Na₂EDTA·2H₂0, 9g of soluble starch, 1g of yeast extract, 8g of agar and 1000mL of water, and the pH value is 6.0.

Surface disinfection: soil and attachments on roots and tubers of wild bletilla striata plants are gently shaken off, washed under running water, and the cleaned roots and tubers of the bletilla striata plants are placed on filter paper to absorb water for later use. On a superclean workbench, the rhizoma bletillae roots and tubers are placed in sterile water, fully shaken, cleaned and fished out, surface disinfection is carried out in sequence, surface disinfection is carried out for 15s in 75% alcohol, disinfection is carried out for 6-10min in 2% sodium hypochlorite solution, and the sterile water is washed for 4-5 times. The common tissue block separation method is adopted, and the rhizoma bletillae roots and tubers with the sterilized tissue surfaces are placed on sterile filter paper and are dried by suction.

Separating and purifying strains: cutting disinfected rhizoma bletilla root and tuber into small blocks of 1cm × 1cm, inoculating root and tuber tissue blocks on PDA plate culture medium, inoculating 4 tissue blocks in each culture dish, placing in a mold incubator, keeping constant temperature at 28 deg.C, dark, and inversely culturing. In order to ensure that the bletilla striata roots and tubers indicate complete sterilization, sterile water for cleaning the bletilla striata roots and tubers for the last time is taken by a sterile liquid transfer gun and coated on a new PDA culture medium, 3 groups of culture dishes are repeated and placed in a mould incubator for inverted culture for 7d at 28 ℃, if no foreign bacteria grow on a PDA flat plate, the tissue surface is proved to be completely sterilized, and the separated fungi are endophytic fungi. After the tuber tissues of the rhizoma bletillae are cultured on the PDA culture medium for 2-3 days, hyphae can be observed to grow out from the edges of the tissue blocks, when the hyphae grow to colonies visible to the naked eye, a small amount of hyphae are picked from the edges of the colonies by using a sterile toothpick and inoculated on a new PDA culture medium, and the inoculation is repeated until pure colonies are obtained.

Inoculating the separated endophytic fungi into a PDA plate, and culturing at 28 ℃ for 7-10 days for later use.

1.2 screening

Adopting the symbiotic culture of the bletilla aseptic seedlings and the endophytic fungi to carry out primary screening on the growth promoting activity of tested bletilla striata endophytic fungi.

Preparing symbiotic culture medium in advance according to the formula of the DE culture medium, and subpackaging into 250mL tissue culture bottles, wherein each bottle is sterilized at 121 ℃ for 30min for later use. Taking out the bletilla striata aseptic tissue culture seedlings on an ultraclean workbench, washing the culture medium carried out from the roots with sterile water, placing the culture medium on sterile filter paper for moisture absorption, and then transferring the culture medium into a DE culture medium. 4 plants are inoculated in each bottle and arranged in a central shape like a Chinese character 'kou', and the bottles are placed in a culture room for culture for 15 days after the inoculation is finished, the temperature is 25-26 ℃, the illumination is 8-12 hours every day, and the illumination intensity is 2000-3000 Lux. After the bletilla striata aseptic seedlings grow on the DE culture medium for 15 days, the growth state of the bletilla striata aseptic seedlings is basically stable. At the moment, fungus cakes with the diameter of 0.6cm are respectively taken from the edges of activated bletilla striata endophytic fungi mother colonies as inoculation materials, the fungus cakes are selected to be inoculated into the middle of 4 bletilla striata seedlings, the distance between the seedlings is about 2cm, each tissue culture bottle is inoculated with one fungus cake, each strain is inoculated into 3 repeated bottles, and a control group is inoculated into a sterile PDA agar block. Sealing after inoculation, placing in a culture chamber for culture at 25-26 deg.C under illumination intensity of 2000-. The growth states of the fungi and the rhizoma bletillae tissue culture seedlings are regularly observed. After 30 days of symbiotic culture, selecting out fungi which can be well symbiotic with the bletilla aseptic seedlings by taking the moderate growth speed of the fungi and the normal growth of the bletilla aseptic seedlings as standards. The strains obtained from the preliminary screening were re-inoculated in DE medium in the same manner, 3 sterile seedlings per bottle, and inoculated into sterile agar blocks of the same size as a control. After symbiotic culture for 60 days under the same culture conditions, taking out the control seedlings and the symbiotic seedlings respectively, washing residual culture medium at roots, and sucking surface water by using filter paper. The plant height, the number of new roots, the number of new buds and the diameter of tubers are measured.

As a result, 5 growth-promoting fungus strains with strong white and growth-promoting effects are obtained, wherein one of the 5 growth-promoting fungus strains is named as 1-G1.

1.3 identification

(I) morphological characteristics of the Strain

And (3) observing morphological characteristics of thalli: endophyte fungal strains 1-G1 to be identified were inoculated on PDA medium, cultured at 28 ℃ and observed for culture characteristics and color changes at 5, 14 and 20 days, respectively. And taking the color characteristic of stable maturity as the culture characteristic of the strain, and taking the culture characteristic as the basis of identification. The color of aerial hyphae, the size, color, tissue shape, surface shape of colony, etc. are recorded as reference characteristics.

As shown in fig. 1, the colony morphology characteristics: on the PDA culture medium, the colony is circular, and the hyphae in the culture medium are gray black, radial, and the aerial hyphae are gray black and cotton-like.

(II) Strain ITS sequence and phylogenetic analysis thereof

Preparation of DNA template:

reagent: (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) mixture, isopropanol, ddH 2O;

DNA extraction:

an appropriate amount of fungal hyphae was taken, 600 μ L of CTAB extraction buffer (20mmol/L Tris (pH 8.0), 3% CTAB, 1.4mol/L NaCl, 10% DTT) was added, a little sterilized quartz sand was added, and the hyphae were ground thoroughly. The slurry was transferred into a 1.5mL EP tube, and the EP tube was shaken every 10min for 30min during a water bath at 65 ℃ to mix the slurry. After water bath at 65 ℃ and ice bath for 10min, 400 mu L of a saturated phenol-chloroform-isoamyl alcohol (25:24:1) mixture is added and mixed evenly. Centrifuge at 12000rpm for 10 min. And adding 400 mu L of supernatant into precooled isopropanol, uniformly mixing, standing, carrying out ice bath for 1 hour, centrifuging at 12000rpm for 10min, removing the supernatant, drying in the air, carrying out heavy suspension by using 20 mu L of ddH2O, and taking 2 mu L of the supernatant as a PCR template to amplify the ITS sequence.

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

(2) An amplification primer: 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) An amplification system: the ITS sequence PCR amplification system is shown in Table 1.

TABLE 1 ITS sequences PCR amplification System

Reactants	Sample addition amount
		2X TagMasterMix	25μL
Primer-1	1μL
		Primer-2	1μL
Template DNA	2μL
		ddH₂O	21μL
Total volume of reaction	50μL

The PCR reaction conditions are shown in Table 2.

TABLE 2 PCR reaction conditions

Note: step 2 was performed for 30 cycles.

And (3) electrophoretic detection of PCR amplification products:

the electrophoresis conditions were 1% agarose gel ((containing Super GelRed 10000X aqueous solution 1. mu.L/10 mL), 1X TBE electrophoresis buffer, electrophoresis at 90V for 1 hour, Loading 4. mu.L PCR product, mixing with 1. mu.L Loading dye, and spotting.

The results were observed under a gel imaging system UV lamp, and the length of the amplified fragment was determined using DL1000DNA Marker from TaKaRa as a nucleic acid standard molecular weight reference. The amplification product band should be at the position of the standard 400-700 bp.

PCR product purification and sequencing: is carried out by Shenzhen Hua Dagen science and technology Limited.

And (3) construction of a phylogenetic tree:

and (3) logging in NCBI, comparing the fungal rDNA ITS sequence obtained by sequencing with a sequence in a GenBank database, recording the login number, the most similar strain, the similarity and the like, downloading the rDNA ITS sequence of the strain, and performing cluster analysis and constructing an NJ phylogenetic tree by using MEGA 7. The ITS sequence of 1-G1 is shown in SEQ ID No. 1.

Performing Blast homology comparison on the rDNA ITS sequence in GeneBank to obtain a representative strain with highest similarity, recording the registration number, the most similar strain, the similarity and the like, downloading the rDNA ITS sequence of the strain, and constructing an NJ phylogenetic tree (as shown in figure 4). The strains with the highest similarity to strains 1-G1 are shown in Table 3.

TABLE 3 strains with closest similarity to fungus 1-G1

A500-and 600-bp fragment was amplified from the genomic DNA of the strain using primers ITS1 and ITS4, sequenced and aligned by BLASTn in GenBank.

The results showed that strain 1-G1 has very high base sequence similarity to the fungus in the genus Diaporthe, so the reference strain sequence of Diaporthe in GenBank was downloaded for phylogenetic analysis, and Diaporthe sp. (KX110392) in Diaporthe was used as the outer population. In the constructed phylogenetic tree, multiple strains of 1-G1 and Diaporthe sp. were clustered together to form terminal branches supporting 99% strength.

The base similarity comparison result shows that the base sequences of 1-G1 and Diaporthe sp are not different, and the sequence similarity is 100%. The strain was preliminarily identified as Diaporthe sp, integrating morphological and molecular biological characteristics.

Example 2: detection of growth promoting characteristics of active strains

2.1 detection of Indolylacetic acid (IAA) synthesizing ability of active strain 1-G1 fermentation liquor

Accurately weighing 1mg IAA, and dissolving in 10mL distilled water to obtain 100 μ g/mL IAA control solution. The gradient diluted IAA control solution was 10, 20, 30, 40, 50, 60, 70, 80 ug/mL. Mixing the IAA solution and the Salkowski colorimetric solution according to the volume ratio of 1:1, standing at room temperature in a dark place for 30min, and measuring the OD530 of the IAA solution with each concentration. A standard curve is drawn by taking the concentration of IAA as an abscissa and the OD530 of the IAA solution at each concentration as an ordinate.

20mL of IAA detection medium was added to a 50mL sterilizable centrifuge tube, sterilized at 121 ℃ for 20min, and then cooled for future use. On a clean bench, a punch is used for punching a 0.6cm mushroom block at the edge of the activated mother mushroom colony, the mushroom block is respectively inoculated into the centrifuge tube by using a sterile toothpick, two bacteria are inoculated into each test tube, 3 tubes are repeated, and a sterile agar block with the same size is inoculated to serve as a blank control. Culturing at 150r/min for 5 days at 28 deg.C in a shaking table. Dropping 100 μ L of culture solution onto white ceramic plate, adding 100 μ L Salkowski colorimetric solution, standing the white ceramic plate at room temperature in dark place for 30min, observing color change, adding 100 μ L sterile culture medium into colorimetric solution as control, and adding 50mg/L IAA and 25mg/L IAA into positive control colorimetric solution. And centrifuging 8000r/min of a strain culture solution with obvious color change for 10min, respectively taking 2mL of supernate, filtering the supernate through a 0.22 mu m microporous filter membrane, filtering and sterilizing, adding 2mL of Salkowski colorimetric solution, standing the mixture at room temperature in a dark place for 30min, performing the same treatment on a blank IAA detection culture medium to serve as a control, adjusting zero, measuring OD530 of each mixed solution, and calculating the IAA content of the sample by referring to the obtained IAA standard curve, wherein the unit of the IAA content is mu g/mL, and 3 repeats.

As shown in FIG. 2, 100. mu.L Talkowski colorimetric solution was added to 100. mu.L IAA solution and 25. mu.L IAA solution in the left positive control, respectively, and after standing in the shade for 30min, the mixture turned colorless to pink, and the color became darker as the IAA concentration became higher. And (3) centrifuging the fermentation liquor of the fungus 1-N2, taking the supernatant, mixing the supernatant with Salkowski colorimetric solution, and standing for 30min in the shade to ensure that the mixed solution has obvious pink color change.

IAA content determination is carried out on fermentation liquor of fungi 1-G1, and the IAA content is 42.38 mug/mL. The growth promotion effect of the IAA is mainly to promote the growth of cells, particularly the elongation of the cells, which can promote the rooting of cutting slips. The fungus 1-G1 shows obvious growth promoting activity in the bacterin symbiosis and pot experiment, and the strain 1-G1 has strong capability of synthesizing IAA, and the result accords with the test result of early fungus growth promotion.

2.2 detection of synthetic ability of Strain siderophore

Adding 20mL of iron-free Chachi culture medium into a 50mL of sterilizable centrifuge tube, sterilizing at 121 ℃ for 20min, taking out and cooling for later use. On a clean bench, a punch is used to punch 0.6cm of fungus blocks on the edges of activated mother colonies, the fungus blocks are respectively inoculated into the centrifuge tube, two fungus blocks are inoculated into each test tube, each fungus block is repeated for 3 tubes, sterile agar blocks with the same size are added into a control, and the control is cultured for 5 days at the temperature of 28 ℃ and at the speed of 150 r/min. Centrifuging the culture solution at 8000rpm for 10min, collecting supernatant, placing the supernatant and CAS color development solution on a white ceramic plate according to a volume ratio of 1:1, shaking gently, mixing, observing color change, recording the color change of the mixed solution to red, purple or yellow culture solution, and taking a picture for recording. The strain culture solution with the color changed into red, purple or yellow is centrifuged for 10min (8000rpm), 2mL of supernatant is respectively filtered through a 0.22-micron microporous filter membrane, after filtration and sterilization, 2mL of CAS colorimetric solution is added, after standing at room temperature for 1h, the solution is zeroed by distilled water, the OD680 of the strain culture solution is measured and is counted As 'As', 2mL of sterile iron-free Chachi culture solution is taken, and the OD680 of the strain culture solution is measured As a reference value and is counted As 'Ar' according to the same method. The change of the blank OD680 value determines the concentration of the siderophore in the culture solution. The concentration of the siderophore in the test was expressed As siderophore activity unit (SU) ═ Ar [ (Ar-As)/Ar ] × 100%. The larger the SU value is, the stronger the ability to synthesize siderophore, and the smaller the As/Ar is, the higher the siderophore content in the culture broth. Generally, the As/Ar of the bacteria with the capability of high-yielding siderophore is less than 0.5.

In nature, the iron element exists in the form of a complex of FeO and iron hydroxide, and this insoluble state is very difficult to be utilized by living beings. Siderophores are a class of small molecule metal ion chelates produced by microorganisms to Fe3⁺Has strong affinity.

As shown in figure 3, in 5 endophytic fungi, the suspension of fungi 1-G1 is mixed with CAS color development solution to react in red, and the color of the sterile culture solution is light green after being mixed with CAS. Indicating that fungus 1-G1 has the ability to synthesize siderophores.

TABLE 4 quantitative determination of siderophores produced by the strains

As shown in Table 4, strain 1-G1 had SU 0.5539 and As/Ar less than 0.5, indicating that the strain is a siderophore strain.

In conclusion, the strain bletilla striata and endophytic fungi 1-G1 have strong IAA production capacity and siderophore synthesis capacity. In addition, the seedling growth promoting agent plays a role in obviously promoting growth in a seedling growth promoting test. Therefore, the strain bletilla striata endophytic fungus 1-G1 fermentation liquor has obvious potential in the aspect of ecological planting of bletilla striata.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

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Claims

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.