CN111925945B - A tobacco pipe fungus G14 and its application - Google Patents

Abstract

The invention discloses a strain of Bjerkandera adusta G14 and its application. Bjerkandera adusta G14 was preserved in the General Microorganism Center of China Committee for the Collection of Microbial Cultures on July 6, 2020, and the preservation number is CGMCC No. 20218. The tobacco pipe fungus G14 of the present invention has various application functions, has good functions of biocontrol, growth promotion and decolorization, and has high development and utilization value for organisms and environment.

Description

A tobacco pipe fungus G14 and its application

technical field

The invention belongs to the technical field of environmental microbes, and relates to a pipe fungus G14 and its application.

Background technique

Plant endophytic fungi (Plant endophytic fungi) commonly exist in healthy plant tissues, and refer to those that spend a certain stage or all stages of their life history in various tissues and organs of healthy plants, and the host does not show any symptoms. Fungioid. It is considered to be one of the important sources of biological control factors for plant diseases. It can directly face the infection of some pathogenic bacteria, attack the pathogen itself or the pathogenic factor to degrade it, produce antagonistic substances, or induce the plant itself to produce systemic resistance to suppress pathogens, etc., while the genes of the plant itself still maintain natural traits , is a natural resource bank for biological control of plant diseases. Endophytic fungi exist in most plants and are rich in resources. They have the characteristics of wide distribution and many types. There are many kinds of secondary metabolites. Terpenes and their saponins, alkaloids, and aromatics can be isolated from endophytic fungi. The structural types of antibacterial substances such as steroids, steroids, and peptides are a hot spot in the development of antibacterial drugs. They have the functions of inhibiting the growth of pathogenic bacteria, improving plant resistance, promoting plant growth, and degrading chemical pollutants. They are widely used in agriculture, forestry, medicine and environmental protection.

Blueberry ( Vaccinium spp.) belongs to the genus Vaccinium of Rhododendronaceae, also known as lingonberry, blueberry, etc. It is a fruit tree of perennial shrubs or small shrubs. Its fruit has a unique flavor, blue in color, beautiful in color, wrapped in white fruit powder, delicate in flesh, sweet and sour, and has a refreshing and pleasant aroma, which is favored by many consumers. Blueberries can be eaten fresh or processed into juice drinks, blueberry wine, etc., and are known as "golden berries" and "the king of fruits in the world". Since the opening of the blueberry market, people have maintained an enthusiastic attitude towards its demand, and the biggest limiting factor affecting the blueberry income is the soil problem. Phosphorus plays an important role in the growth of crops and is one of the essential mineral nutrients for plants. Recently, due to people's unreasonable farming methods, many poor soil environments have occurred, resulting in a serious lack of phosphorus in most crops, which in turn leads to a large reduction in crop yield.

Fusarium oxysporum and Fusarium verticillium can cause blueberry root rot and damage the roots of plants. It is one of the most serious diseases in blueberry production. Alternaria alterniaria can cause blueberry leaf spot, which is serious in the main blueberry producing areas. The main pathogen of pepper anthracnose is Anthracnose oxysporum, which can cause rotten leaves, rotten fruits, and even plant death of peppers. The disease commonly occurs in pepper producing areas in Guizhou. Sclerotinia sclerotiorum is the main pathogenic bacterium of canker sores, which can cause diseases of peanuts, sweet potatoes, apples and other plants. The damage to peanuts mostly occurs in the mature stage of plants. The pathogens can invade the roots or stem bases of peanuts directly or from wounds, causing Rhizomes, fruit stalks and pods are diseased and rotten. Sclerotinia can infect crops and vegetables, and is one of the important diseases of Sclerotinia sclerotiorum, which can reduce the yield of diseased plants by half.

my country's printing and dyeing enterprises continue to develop. Due to the influence of chemical reactions in the production and production process of dyes, in addition to their own raw materials, some additional products will be produced. The two together constitute dye production wastewater. Some untreated or excessive dye wastewater is directly discharged into natural water bodies, which will cause large areas of water pollution. Because of its poor biodegradability and high toxicity, it leads to the death of a large number of aquatic organisms, and causes poor light transmission intensity of water bodies and difficulty in degradation. , and reduced photosynthesis.

At present, most plant endophytic fungi are mainly isolated from the rhizosphere soil, roots, stems, and leaves of plants, and a large number of plant endophytic fungi resources are obtained. Fewer endophytic fungi were isolated and identified from valuable edible fruits. Moreover, most of the isolated strains are monofunctional, while few strains possess multiple functions.

The problems of pesticide residues and environmental pollution have aroused widespread concern from the public, and green and pollution-free agricultural products are increasingly favored by people. Therefore, it is particularly important to increase the available phosphorus content in the soil to increase the absorption of phosphorus by crops and reduce the use of phosphorus fertilizers; to control plant diseases biologically and reduce the use of chemical pesticides; to degrade dye wastewater greenly.

Contents of the invention

The object of the present invention is to provide a pipe strain G14 and its application. The tobacco pipe fungus G14 of the present invention has various application functions, has good functions of biocontrol, growth promotion and decolorization, and has high development and utilization value for organisms and environment.

Technical solution of the present invention: a strain of Bjerkandera adusta G14, G14 ( Bjerkandera adusta , G14), was preserved in the General Microorganism Center of China Committee for Microbial Culture Collection on July 6, 2020, and the preservation number is CGMCCNo. 20218 .

In the above-mentioned pipe strain G14, the ITS rDNA nucleotide sequence of the pipe strain G14 is shown in SEQ ID NO.1.

In the above-mentioned tobacco tube fungus G14, the application of the tobacco tube fungus G14 in biocontrol.

In the above-mentioned tobacco pipe fungus G14, the biocontrol is: the tobacco pipe fungus G14 exhibits antibacterial activity against plant pathogenic bacteria.

In the aforementioned pipe fungus G14, the plant pathogenic bacteria include blueberry root rot pathogen ( Fusarium oxysporum ), blueberry root rot pathogen ( F. verticillioide ), blueberry leaf spot pathogen ( Alternaria alternata ), pepper anthracnose pathogen ( Colletotrichum scovillei ), Sclerotium rolfsii , and Sclerotinia sclerotiorum .

Among the above-mentioned fungi G14, the use of the fungus G14 in promoting plant growth.

Among the above-mentioned fungi G14, the growth-promoting plants of the fungus G14 include cabbage and corn.

In the above-mentioned pipe strain G14, the application of the pipe strain G14 in degrading dyes.

In the above-mentioned pipe fungus G14, the degraded dye is as follows: the tobacco pipe fungus G14 secretes a lignin degrading enzyme to decolorize the dye.

In the aforesaid S. fumigatus G14, the lignin degrading enzymes include laccase, lignin peroxidase and manganese peroxidase.

Compared with the prior art, the present invention has the following beneficial effects: the present invention starts from blueberry endophytes, and through targeted screening, obtains a plant with good phosphorus-dissolving function and simultaneous Bjerkandera adusta, a multifunctional endophytic strain with antagonistic and degradative functions, was identified as Bjerkandera adusta by morphological and molecular biology, and was preserved in the General Microorganism Center of China Committee for the Collection of Microbial Cultures. The strain was effective against two blueberry root rot pathogens ( Fusarium oxysporum ), ( F. verticillioide ), blueberry leaf spot pathogen ( Alternaria alternata ), pepper anthracnose pathogen ( Colletotrichum scovillei ), white silkworm pathogen ( Sclerotium rolfsii ) and nuclear Six kinds of pathogenic bacteria of Sclerotinia sclerotiorum exhibit antibacterial activity and biocontrol function, and have strong development and utilization value in the prevention and treatment of blueberry root rot. In addition, it also has a strong phosphorus-dissolving ability, which can promote the growth of cabbage and corn, and has the function of promoting growth. It provides a reference for the development of safe and effective soil phosphorus-dissolving agents by integrating various factors. At the same time, the invention can also secrete three kinds of lignin degrading enzymes, laccase, lignin peroxidase and manganese peroxidase, and has the decolorization function for various dyes, laying a foundation for the application in dye wastewater treatment. Generally speaking, the strain of the present invention is a kind of endophytic strain, has multiple functions, and has high development and utilization value for human beings, organisms and the environment.

Description of drawings

Fig. 1 is the colony morphology diagram of bacterial strain G14; Wherein A is the front side of the colony morphology diagram; B is the back side of the colony morphology diagram;

Fig. 2 is the phylogenetic tree of bacterial strain G14 ITS sequence;

Fig. 3 is the inhibitory effect of bacterial strain G14 to 6 kinds of pathogenic bacteria;

Figure 4 is the phosphorus-dissolving effects of strain G14 in PKO (left) and Montina (right);

Figure 5 shows the growth-promoting effect of strain G14 on Chinese cabbage (left: plant height; right: root)

Fig. 6 is the activity change of laccase, lignin peroxidase, manganese peroxidase in bacterial strain G14 liquid culture process;

Figure 7 shows the decolorization of 5 dyes by the crude enzyme solution of the tuberculosis G14 manganese peroxidase of strain G14.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments, but not as a basis for limiting the present invention.

Example 1: Isolation, Identification and Preservation of Bjerkandera adusta (G14)

1.1 Medium and its preparation

PD medium: peel 200g of fresh potatoes, boil 1.1L of distilled water for 30min, filter through 4 layers of gauze to obtain potato liquid, add 0.05g of chloramphenicol to the potato liquid, and distilled water to dissolve to 1L;

PDB medium: add 20g/L glucose to PD medium;

PDA medium: add 20g/L agar powder to PDB medium;

Carbon source screening medium: on the basis of PD medium, 20g/L carbon source (glucose, maltose, sucrose, soluble starch, fructose) is added, and the pH is naturally added;

Nitrogen source screening medium: based on PDB medium, 1g/L nitrogen source (ammonium nitrate, ammonium chloride, ammonium sulfate, urea, peptone), natural pH;

、MnS

、CaC

、MgS

、ZnS

和FeC

），pH自然。Metal ion screening medium: on the basis of PDB medium, add 0.5mmol/L metal source (CuS

, MnS

, CaC

, MgS

, ZnS

and FeC

), pH natural.

PKO inorganic phosphorus medium: Ca ₃ (PO4) ₂ 3.0 g, sucrose 10.0 g, NaCl 0.5 g, KCl 0.2 g, (NH ₄ ) 2 SO ₄ 0.1 g, MgSO ₄ 7H2O 0.1 g, MnSO ₄ 0.004 g, yeast Cream 0.5 g, FeSO ₄ 0.004 g, distilled water 1 000 mL, agar 15.0 g, pH 7.0.

Montkina organophosphate medium: 10g, (NH ₄ ) ² SO ₄ 0.5 g, NaCl 0.3 g, KCl 0.3 g, lecithin 0.2 g, MgSO ₄ •7H ₂ O 0.3 g, FeSO ₄ •7H ₂ O 0.03g , MnSO ₄ •4H ₂ O 0.03 g, CaCO ₃ 5 g, yeast extract 0.4 g, agar 20 g, distilled water 1 000 mL, pH 7.0.

1.2 Isolation and purification of strains

Using the ultra-clean workbench in the laboratory, soak the collected blueberry fruit with strain G14 in 75% alcohol solution for 10 seconds, then transfer it to 3% (V/V) NaClO solution for disinfection for 3 minutes, and first use sterile Rinse with water and then dry on sterile absorbent paper. Cut the pericarp with a diameter of about 1 cm and inoculate it in PDA solid medium, culture it in a constant temperature incubator at 28°C for 4 days, inoculate the fresh hyphae on the edge of the pericarp into a new PDA medium for cultivation, and transfer it several times to obtain Purified strain G14.

1.3 Identification of strains

Morphological identification of strains: In a clean bench, use a sterile puncher with a diameter of 6.0mm to intercept fresh strain G14 bacterial blocks, put them in the center of the PDA medium, and cultivate them at a constant temperature of 28°C. Observe the growth status, shape, color, etc. of the colony in time, and record the horizontal and vertical diameters of the colony. On the 4th day of culture, the hyphae at the edge of the colony were picked and placed on a glass slide, and the morphological characteristics of the mycelia were observed with an optical microscope for morphological identification. For morphological identification, refer to the "Handbook of Fungal Identification". During the culture period of strain G14, the colony hyphae expanded rapidly on the 2d and 4th day of culture, and the strain covered the culture dish on the 4th day. The colony is characterized by a round shape with ring-shaped edges and a milky white cotton-wool-like center with well-developed hyphae growing radially (Figure 1).

Molecular biological identification of the strain: the hyphae of the newly activated strain G14 were scraped, the genomic DNA of the strain G14 was extracted using the Fungal DNA Midi Kit fungal DNA extraction kit as a template, and the strain was amplified by PCR using fungal universal primers. The amplified PCR product was sent to Chongqing Qingke Xingye Biotechnology Co., Ltd. for sequencing. At the same time, the sequence was compared with the B.adusta sequence from different sources and the ITS sequence of the same and different species in the BlastN comparison results. ClustalW in MEGA7.0 software was used for multiple sequence alignment, and then the phylogenetic tree was constructed using the Neighbor joining method (Neighbor joining, NJ) in MEGA7.0. After the total DNA of strain G14 was amplified by ITS1 and ITS4 primers, the PCR product was sent to Chongqing Qingke Xingye Biotechnology Co., Ltd. for sequencing to obtain a 570bp sequence. After obtaining the sequencing results, log into GenBank and obtain the accession number MT601950. At the same time, the Blast in NCBI was used to compare the similarity of the sequences, and the MEGA7.0 software was used to construct a phylogenetic tree with similar strains (Figure 2). The results showed that the strain B. adusta KJ668570. 1 has the highest similarity and the closest kinship.

1.4 Culture preservation of strain G14:

Preservation unit: General Microbiology Center (CGMCC), China Committee for the Collection of Microbial Cultures, Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, named: Bjerkandera adusta , G14 ), the deposit number is CGMCC No. 20218, and the deposit date: July 06, 2020.

Example 2: Determination of antibacterial properties of tobacco pipe bacteria ( Bjerkandera adusta , G14)

The plate confrontation method was used to determine the antibacterial activity of the strain against six kinds of plant pathogenic bacteria tested. First, all endophytic fungal strains and pathogenic bacteria are activated, and then in the ultra-clean workbench, punch holes on the fresh mycelium of the endophytic fungal strains and pathogenic bacteria edges activated in the PDA plate with a hole puncher with a diameter of 6mm, and respectively punch The bacteria cake obtained from the hole was inoculated on a PDA plate with a diameter of 90 mm. The inoculation point was on both sides of the culture medium, on the same diameter 20 mm from the edge of the plate. Three repetitions were set as the experimental group, and only the pathogenic bacteria were inoculated on the right side of the plate as the control group. Three repetitions were also set up, and then both the experimental group and the control group were placed in a constant temperature incubator at 28°C for cultivation, first placed on the front side, and then placed upside down after 1 day. After 5-7 days, when the pathogenic bacteria colony of the control group grows to about two-thirds of the petri dish, take pictures and record the colony diameter of the pathogenic bacteria in the control group and the colony diameter of the pathogenic bacteria in the experimental group. Inhibition rate calculation: Inhibition rate (%) = ((control group phytopathogen colony diameter-6mm)-(experimental phytopathogen colony diameter-6mm)/(control group phytopathogen colony diameter-6mm))×100. The inhibition rate of strain G14 to two blueberry root rot pathogens ( Fusarium oxysporum ) and ( F. verticillioide ) was 60.84% and 55.69%. The inhibition rate to blueberry leaf spot pathogen ( Alternaria alternata ) was 44.44%. The inhibition rates against Sclerotium rolfsii , Colletotrichum scovillei and Sclerotinia sclerotiorum were 56.92%, 45.71% and 45.45%, respectively. The inhibition rate of strain G14 to 6 kinds of pathogenic bacteria is shown in Fig. 3 .

Example 3: Determination of growth-promoting properties of tobacco pipe bacteria ( Bjerkandera adusta , G14)

Phosphate Solubilization Screening of Endophytic Fungi:

The isolated and purified blueberry endophytic fungi were activated and cultured, and the activated endophytic fungal blocks (d=0.6cm) were picked and inoculated on the PKO inorganic phosphorus medium and Montina organic phosphorus medium respectively, and the inoculation After the final disposable culture dish was placed in a constant temperature incubator at 28°C for 72 hours, it was determined whether the strain had phosphorus-dissolving effect by observing whether the transparent phosphorus-dissolving circle appeared or not, and at the same time measured and calculated the diameter of the phosphorus-dissolving circle D of the strain. Value and the ratio of the colony diameter d value, preliminarily judge whether the strain has phosphorus-solubilizing ability. The experiment shows that the D/d value of G14 in Montina medium is up to 1.7, which shows better phosphorus-dissolving characteristics. In the PKO medium, the D/d value of G14 was 1.7 at the maximum, and the growth rate and the size of the phosphate-dissolving circle of the strain were different in the PKO and Montina medium. The phosphorus-dissolving effect diagram of strain G14 is shown in Figure 4.

The growth-promoting effect of blueberry phosphorus-soluble endophytic fungi on different crops: commercially available corn, cabbage, and wheat were selected as test materials, and the soil under the Pinus massoniana forest in Chongde Building of Guizhou University was used as test soil. Commercially available cabbage, cabbage, and Wheat and corn seeds were sterilized with 3% sodium hypochlorite for 5 minutes, rinsed with sterile water, germinated in the dark at 28°C, and sowed in the seedbed. After 5 days, the seedlings with basically the same growth were selected for the experiment. The screened phosphate-dissolving strains were fermented in PDA medium at a shaker speed of 180 r/min and cultured at 28°C for 7 days to prepare a bacterial suspension. One control group and two treatment groups were set up for each plant, and each group had 3 replicates, and each replicate treated 10 plants. The root irrigation method is adopted, and the bacterial suspension is placed in the seedling pot. The soil was kept moist throughout the experiment, and the plants were harvested after 30 days, and the observations of plant height, fresh weight, root system and leaf area were measured. During the actual test, it was observed that the strain G14 had no obvious growth-promoting effect on the plant height of corn and wheat, but had a certain growth-promoting effect on the roots of corn, and had an obvious growth-promoting effect on cabbage, as shown in Figure 5.

Example 4: Decolorization Characteristic Determination of Bjerkandera adusta (G14)

Determination of Laccase, Lignin Peroxidase and Manganese Peroxidase Activities of Tobacillus fumigatus G14

Put 70mL of PDB into a 250mL Erlenmeyer flask, sterilize at 121°C for 30 minutes, put 4 pieces of new activated mycelium with a diameter of 9mm into each Erlenmeyer flask on an ultra-static workbench, and place them at a constant temperature of 28°C for cultivation Cultivate in a box protected from light, absorb 1.0mL fermentation broth on the 2nd, 4th, 6th, 8th, 10th, and 15th day after inoculation, centrifuge at 7000r/min for 5min, take the supernatant and measure the enzyme activity with a UV-visible spectrophotometer.

Laccase (Lac) activity assay: 3.0mL reaction system contains 1.95mL citric acid-disodium hydrogen phosphate buffer (pH5.0), 1.0mLABTS (1.0mmol/L), 0.05mL supernatant fermentation broth, 30℃ water bath After 3 minutes, the change of absorbance at 420 nm within 3 minutes was measured. An enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the conversion of 1 μmol ABTS substrate into product per minute [ABTS: ε ₄₂₀ =3.6×10 ⁴ L/(mol·cm)]. Each of the above treatments was repeated 3 times.

Determination of manganese peroxidase (manganese peroxidase) activity: 0.84mL sodium malonate (0.05mol/L), 0.05mL MnSO ₄ (0.01mol/L), 0.05mL2,6-dimethoxyphenol (2,6-DMP) (0.01mol/L), 0.05mL supernatant fermentation liquid, 0.01mL H ₂ O ₂ (0.01mol/L) were loaded into a cuvette, distilled water was used as a control group, and the absorbance at 468nm was measured at change within 1 min. One enzyme activity unit (U) is defined as the amount of enzyme that can catalyze the conversion of 1 μmol2,6-DMP substrate into the required amount per minute. [2,6-DMP: ε ₄₆₈ =4.96×10 ⁴ L/(mol·cm)]. Each of the above treatments was repeated 3 times.

Determination of lignin peroxidase (LiP) activity: mix 1.7mL sodium potassium tartrate tetrahydrate solution (0.25mol/L) and 0.05mL veratrol (VA) solution (0.1mol/L) solution, and place in a water bath at 30°C Preheat the pot, then add 0.05mL supernatant fermentation broth, and finally add 0.05mL H ₂ O ₂ (0.1mol/L), and measure the change of absorbance at 310nm within 3 minutes ^[26] . An enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the conversion of 1 μmol VA substrate per minute [VA: ε ₃₁₀ =9.3×10 ³ L/(mol·cm)]. Each of the above treatments was repeated 3 times.

The activities of three lignin-degrading enzymes produced by strain G14 were monitored from the second day, and the activities of the three enzymes increased with the extension of culture time. Among them, the laccase activity reached a peak of 1.67U/L on the 8th day, and then gradually decreased; the manganese peroxidase activity also reached a peak of 12.5U/L on the 6th day, and then gradually decreased; lignin peroxidation Biozyme activity decreased gradually after reaching the maximum value of 4.38U/L on the 6th day. Figure 6 shows the change of enzyme production activity of Nicotiana fumigatus G14 over time.

Decolorization of 5 kinds of dyes by the crude enzyme solution of manganese peroxidase of G14 tuberculosis: Congo red, malachite green, crystal violet, chrome black T and methyl orange were added to the PDB medium respectively to make the dyes The final concentration is 50mg/L. After being sterilized under high temperature and high pressure at 121°C for 30 minutes, under aseptic conditions, insert 4 pieces of newly activated tobacco pipe bacteria G14 with a diameter of 1 cm into each flask. The inoculated Erlenmeyer flask was cultured in a constant temperature incubator at 28°C in the dark, and 1 mL of fermentation broth was drawn when the enzyme production of Nicotiana tabacum G14 was relatively large, and the dye fermentation was detected at the maximum absorption wavelength of different dyes using a UV-visible spectrophotometer. The absorbance value of the solution was compared with the uninoculated dye culture solution as a control. Use a UV spectrophotometer to scan the maximum absorption wavelength of the dye in the range of 300–800 nm. The formula for calculating the dye decolorization rate is as follows. The calculation method of the dye decolorization rate is as follows: R=(A0-A1)/A0×100% R is the decolorization rate, A0 is the absorbance value of the control, and A1 is the absorbance value under the treatment conditions.

The crude enzyme solution of manganese peroxidase from G14 tuberculosis can decolorize the five dyes, as shown in Figure 7, but the decolorization effects are different, and the decolorization ability of the dyes is ranked as Congo red > Nuo black T > methyl orange > crystal violet > Malachite green. For this different decolorization effect on different dyes, the analysis is due to the different chemical composition of the dyes.

sequence listing

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Claims (1)

1. A strain of Bjerkandera adusta G14, characterized in that: Bjerkandera adusta G14 was deposited in the General Microbiology Center of China Committee for the Collection of Microorganisms on July 6, 2020, and the preservation number is CGMCC No.20218 .

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