CN106701599A - Screening method and identification method for mycorrhizal fungus for promoting selenium enriching of vaccinium uliginosum and application thereof - Google Patents

Abstract

The invention discloses a screening method and identification method for a mycorrhizal fungus for promoting selenium enriching of vaccinium uliginosum and an application thereof. The strain is isolated from vaccinium uliginosum plant roots, a candidate strain is screened through analysis of the vaccinium uliginosum fruit organic selenium content, fruit nutritive element content, mycorrhizal colonization and other test data, and then the fungus is identified to belong to mucoraceae strains by using morphology and molecular biology methods. The strain solves the defect that vaccinium uliginosum cultivation acidic soil is not conducive to absorption of inorganic selenium, is easy to culture and low in cost, can be used for preparation of a mycorrhizal fungus preparation, is used for commercial production of the vaccinium uliginosum, improves the nutritive element absorption of the vaccinium uliginosum, and improves a vaccinium uliginosum root system microbial environment.

Description

A screening method and identification method of mycorrhizal fungi that promote blueberry selenium enrichment and its application

technical field

The invention belongs to the field of strain identification and application, and relates to a screening method of mycorrhizal fungi that promote selenium-enrichment of blueberry fruit, an identification method and an application thereof.

Background technique

Blueberry is lingonberry (Vaccinium uliginosum), also known as Dusi, and wild blueberry is called persimmon (Xing'an Mountains, Yichun). It belongs to the genus Vaccinium (Ericaceae) of the Rhododendron family (Ericaceae). It is native to North America and has about 450 species. Blueberries can be divided into four categories: lowbush, half-highbush, highbush and rabbiteye blueberries. Blueberry fruit is rich in arashin, anthocyanins, flavonoids and other polyphenolic physiologically active ingredients with antioxidant properties, known as the "king of berries". The cultivation soil of blueberries is acidic soil (PH4.0～5.0), the roots are thin and lack root hairs, the contact area is small, the distribution is shallow, and the absorption capacity is weak. Under natural conditions, mycorrhizal fungi can form a mutually beneficial symbiotic relationship with plants, so the screening of beneficial mycorrhizal fungi can promote the absorption of nutrient elements of rabbit-eye blueberries, help blueberries grow and improve fruit nutritional value, and improve blueberry root microorganisms environment and enhance blueberry growth vigor and resistance to diseases and insect pests.

Selenium is one of the essential trace elements in the human body, and its physiological function is mainly manifested in the antioxidant effect of selenoproteins and selenoenzymes. China is a large selenium-deficient country. There is a low-selenium zone in 72% of China's land area, 30% of which are severely selenium-deficient areas, and natural foods such as grains have low selenium content. Large and medium-sized cities such as North China, Northeast China, and Northwest China are all selenium-deficient areas. Many diseases have been successfully prevented and treated in my country through selenium supplementation, such as Keshan disease, Kaschin-Beck disease, cardiovascular disease, liver disease and tumors. The use of biotransformation pathways to produce organic selenium with higher biological activity can reduce the toxicity of inorganic selenium. The blueberry fruit itself is a berry with high nutritional value, and it will have a very broad prospect as a carrier of selenium bioorganization.

Read et al. isolated a mycorrhizal fungus from blueberry plants in 1978. Through its infection characteristics and backgrafting tests, the results showed that mycorrhizal fungi can significantly promote the growth of blueberries. In 2007, Kosola et al. showed that after inoculation of mycorrhizal fungi, blueberry's ability to absorb nitrate nitrogen would be enhanced. In 2010, Liu Yan isolated two strains of mycorrhizal fungi from red bean lingonberry, mixed them and inoculated them on blueberry seedlings, measured them 80 days later, and found that the total dry weight and number of leaves of the seedlings inoculated with mycorrhizal fungi were higher than those of the uninoculated control. 76.1% and 60.7%; at the same time, the absorption of nitrogen and phosphorus in the above-ground and lower parts of blueberry seedlings increased after inoculation, and the inoculation treatment significantly improved the growth of blueberry seedlings. Yuan Jixin et al. isolated 15 strains in 2013 and inoculated them on blueberry seedlings, which showed that they could promote the growth of blueberry seedlings and increase the absorption of nitrogen, phosphorus and potassium elements.

At present, the selenium content of selenium-enriched fruits on the market is 10-50 μg/kg. Blueberry fruit itself has high nutritional value, but the absorption of selenium is affected by the weak root system and acidic soil. Therefore, using mycorrhizal fungi to promote the transformation and absorption of inorganic selenium in blueberry roots is an effective way for blueberry selenium enrichment.

In view of the above analysis, the present invention isolated the mycorrhizal fungus that promotes blueberry selenium enrichment from the blueberry rhizosphere and carried out molecular identification. .

Contents of the invention

The purpose of the present invention is to obtain the mycorrhizal fungus that is beneficial to the selenium-enrichment of blueberry in acidic soil, so as to cultivate the selenium-rich blueberry with high nutritional value, which is used for the commercial production of blueberry, and provides a kind of mycorrhizal fungus that promotes the selenium-enrichment of blueberry fruit Screening method and its identification method and its application.

In order to achieve the above object, the present invention adopts the following technical solutions:

A screening method for mycorrhizal fungi that promotes blueberry selenium enrichment is characterized in that it comprises the following steps:

(1) After disinfecting the collected blueberry mycorrhizae, place them on a PDA solid medium containing antibiotics at pH 6-8, cultivate them in a constant temperature incubator at a temperature of 25 to 30°C, and inoculate the isolated mycorrhizal fungi into Purify and cultivate on PDA medium containing antibiotics, inoculate the purified strains on PDA slant, and store at 3-4°C for later use;

(2) the mycorrhizal fungus obtained in step (1) is returned to blueberry seedlings and plants, and the content of the leaves and fruit nutrients of blueberries is detected by quantum resonance and ICP-MS, and the mycorrhizal fungi that promote the selenium enrichment of blueberries are selected, That's it.

The identification method of the mycorrhizal fungus that promotes blueberry selenium enrichment is characterized in that,

The screened mycorrhizal fungi were identified by molecular biology and morphological methods to determine the species of mycorrhizal fungi.

The identification method of the mycorrhizal fungus that promotes blueberry selenium enrichment is characterized in that,

The molecular biology identification method is as follows: the genome of the screened mycorrhizal fungi is extracted, and the ITS sequence of the fungal rDNA is used as a universal primer for PCR amplification. The sequences obtained by sequencing are compared and analyzed by BLAST in the GenBank database. In the results, sequences with a homology greater than 97% were analyzed using the CLUSTAL-W software for sequence matching, and the Neighbor-joining method in the MEGA6 software was used to construct a phylogenetic tree to determine the phylogenetic status of mycorrhizal fungi.

The identification method of the mycorrhizal fungus that promotes blueberry selenium enrichment is characterized in that,

It was determined that the mycorrhizal fungus beneficial to blueberry's selenium enrichment was Mucorcircinelloides (Code: CANV) of the family Mucoraceae.

The application of the mycorrhizal fungus that promotes blueberry selenium enrichment is characterized in that,

It can be used to prepare mycorrhizal fungus preparations and be applied to the production of blueberries.

The Se content of blueberry fruit inoculated with this strain can reach 0.09-0.16 μg/g, and the content of other nutrients such as Ca, Fe, Zn, Mg, Mn has a certain percentage increase; the survival rate of blueberry seedlings inoculated with this strain, seedling High, leaves N, P, K content also increased correspondingly.

The method for isolating said mycorrhizal fungi is characterized in that step (1) includes the following steps: cutting the collected blueberry mycorrhizal fungi into 3-4 cm root segments, cleaning them with double distilled water, and then placing them in a Use 75% alcohol, 75% alcohol and 0.1% mercuric chloride in a 90mm petri dish to sterilize the surface, cut the sterilized root sample into 0.5cm with sterilized scissors and place it in the PDA medium containing streptomycin Medium culture, the size of the petri dish is 90mm, cultivated in a constant temperature incubator at a temperature of 25-30°C, and cultivated for 3-5 days. A single colony is selected with an inoculation needle and placed on a PDA medium containing streptomycin for purification. The purified The strains were inoculated on PDA slant and stored at 4°C.

The determination of the content of nutrient elements is characterized in that it comprises the following steps: Step (2) adopts inductively coupled plasma mass spectrometry (ICP-MS) to measure the total selenium and nutrient element content of the fruit; N, P, K relative content.

The assay method of described blueberry selenium and nutrient element content is:

Take 0.3g of selenium-enriched blueberry fruit dry powder, add 9-11ml of digestive juice and shake well, the digestive juice is concentrated nitric acid: high hypochlorous acid = 4:1, leave it overnight, place it on a hot plate and heat until organic matter Decompose or the color deepens, add 1~3ml of digestive solution to continue digestion, control the temperature below 200°C, repeat this until the digestive solution is clear, and white smoke of HClO ₄ comes out, continue to heat until the solution is 1~3ml, and use 60% concentrated HCl Set the volume in a 25ml volumetric flask, then add 0.5% NaBH ₄ to the end of the reaction, use ICP-MS, inductively coupled plasma mass spectrometry to measure the content of selenium and nutrient elements.

Described blueberry selenium relative content detection method is:

Put 0.2g of blueberry plant leaves on the resonance plate, measure the frequency and energy of the micro-magnetic field of the sample through the hand-held sensor, amplify the instrument, and compare it with the standard "quantum resonance spectrum" of the nutritional index inside the instrument after computer processing, and output Corresponding relative magnitude values from negative to positive.

The method for morphological identification of mycorrhizal fungi comprises the following steps: screen out the dominant strains that promote blueberry selenium-enrichment according to the detection results of quantum resonance and ICP-MS, conduct preliminary identification of the strains by observing the appearance of colonies and hyphae with optical microscopy, and Comparison of morphological differences using trypan blue-stained blueberry root infection test. See Figure 2. According to the fungal identification manual, the morphological classification was carried out.

The molecular biological identification method of mycorrhizal fungi comprises the following steps: extracting the genome of the screened mycorrhizal fungi, and performing PCR amplification with the ITS sequence of the fungal rDNA as a universal primer. The sequence obtained by sequencing is carried out in the GenBank database. BLAST comparison analysis, using the CLUSTAL-W software for sequence matching analysis on the sequences with more than 97% homology in the search results, using the Neighbor-joining method in the MEGA6 software to construct a phylogenetic tree to determine the phylogenetic growth of mycorrhizal fungi Status, complete molecular identification.

See Figure 1 for the technical circuit of the isolation and identification method of mycorrhizal fungi that promote blueberry selenium enrichment.

Through the above method, we screened out a mycorrhizal fungus that promotes selenium-enrichment in blueberry acidic soil from blueberry rhizosphere microorganisms, and identified it as Mucorcircinelloides by morphological observation and molecular biology methods. See accompanying drawing 2, Fig. 3.

The application of a mycorrhizal fungus that promotes blueberry selenium enrichment can be used to prepare mycorrhizal fungal preparations, inoculate blueberry seedlings to promote their growth; inoculate blueberry plants to increase the content of selenium in their fruits, and other nutritional elements Ca, The content of Fe, Zn, Mn, Cr and other nutrient elements also has a certain percentage increase.

The potted cultivation method of selenium-enriched blueberry seedlings comprises the following steps: putting the cultivation soil into 121° C. high-pressure steam sterilization for 20 minutes, dividing it into 80 g equal portions, pouring them into sterile 10cm×8cm×7cm cultivation pots, and adjusting them with oxalic acid. The pH of the soil is 4.0 to 5.5. Dig a 3cm x 3cm pit with a depth of 5cm, add 2 mushroom cakes of 2cm x 2cm, and move the blueberry seedlings into it. The blueberry seedlings were cultivated in a ventilated and constant temperature (25° C.) light environment, and poured sterile water. After the blueberry seedlings were transplanted and survived, a sodium selenite solution with a concentration of 40 mg/ml was applied to the roots until the selenium content of the soil reached 0.04 mg/kg. The mycorrhizal fungi were inoculated and selenium was applied once every 15 days, for a total of three times.

The method for cultivating selenium-enriched blueberry plants comprises the following steps: uniformly mixing plant ash and soil after autoclaving at 121°C in a ratio of 1:3, adjusting the pH of the soil to 4.0-5.5 with oxalic acid, and adding selenium fertilizer to make the selenium content of the soil reach After 0.04mg/kg, the blueberry plants were transplanted. Inoculate the cultured mycorrhizal fungi, and apply selenium fertilizer at intervals of 15 days until the selenium content in the soil is 0.04 mg/kg. Regular watering (oxalic acid to adjust the pH to 4.5 to 5.5) management.

Beneficial effects of the present invention:

(1) The screening and identification of the present invention to obtain the Mucor circinelloides strain can promote the selenium enrichment of blueberry fruit, improve the nutritional value of blueberry fruit, and reduce the cost of blueberry planting and cultivation management.

(2) The selenium content of blueberries inoculated with mycorrhizal fungi was significantly higher than that of uninoculated blueberries, and the selenium content of selenium-enriched blueberries could reach 0.09-0.163 μg/g

(3) The content of other nutrient elements in blueberries also increased significantly after inoculation: the content of Ca could reach 200-500 μg/g, the content of Zn could reach 1-5 μg/g, the content of Fe could reach 5-30 μg/g, and the content of K could reach The content of Mn can reach 1.5-3.0μg/g, and the content of Cr can reach 0.5-1.5μg/g.

(4) The survival rate of transplanted blueberry seedlings is increased by 20-40%, and the seedling height is increased by 5-10%.

Description of drawings

Fig. 1 is the schematic diagram of the technical circuit of mycorrhizal fungus promoting blueberry selenium enrichment;

Fig. 2 is the picture of Mucor circinelloides fungus of the genus Mucor circinelloides that promotes blueberry selenium;

Figure 3 is a phylogenetic tree diagram of mycorrhizal fungi.

detailed description

Example 1: Cultivation of rabbit-eye blueberry seedlings and inoculation of mycorrhizal fungi: Put the culture soil into 121°C high-pressure steam sterilization for 20 minutes, divide it into 80g equal parts, and pour them into sterile 10cm×8cm×7cm culture pots , use oxalic acid to adjust the soil pH to 4.0-5.5, dig a 3cm x 3cm pit with a depth of 5cm, add 2 cm x 2cm fungus cakes, and move the blueberry seedlings into it. The blueberry seedlings were cultivated in a ventilated and constant temperature (25° C.) light environment, and poured sterile water. After the blueberry seedlings were transplanted and survived, apply sodium selenite solution with a concentration of 40 mg/ml to the roots until the selenium content of the soil reaches 0.04 mg/kg, inoculate the cultured mycorrhizal fungus CANV, and then apply selenium fertilizer once every 15 days for a total of three times . 15 days after the third selenium fertilizer was applied, the leaves of selenium-enriched blueberry seedlings were picked for quantum resonance detection.

After transplanting, the survival rate of the seedlings increased by 38.9%, the height of the seedlings increased by 9.1%, and the contents of selenium, nitrogen, phosphorus and potassium also increased correspondingly according to quantum resonance detection. See Table 1.

Table 1 Detection of relative content of N, P, and K in leaves of selenium-enriched blueberry seedlings inoculated with mycorrhizal fungi

Example 2: Selenium-enriched cultivation of rabbit-eye blueberry plants: Mix the plant ash and soil after autoclaving at 121°C in a ratio of 1:3, adjust the pH of the soil to 4.0-5.5 with oxalic acid, and add selenium fertilizer to make the soil selenium After the content reaches 0.04mg/kg, blueberry plants are transplanted. Selenium fertilizer was applied at intervals of 15 days until the soil selenium content was 0.04mg/kg. Regular watering (oxalic acid to adjust the pH to 4.5 to 5.5) management. After the blueberry plants were transplanted and survived, apply sodium selenite solution with a concentration of 40 mg/ml to the roots until the selenium content of the soil reaches 0.04 mg/kg, inoculate the cultured mycorrhizal fungus CANV, and then apply selenium fertilizer once every 15 days, for a total of three times . Fifteen days after the third selenium fertilizer was applied, the ripe selenium-enriched blueberry fruits were picked for ICP-MS detection. The test results are shown in Table 2.

Table 2. Determination results of selenium and nutrient elements in selenium-enriched blueberries inoculated with mycorrhizal fungi

Claims (5)

1. a screening method of mycorrhizal fungi that promotes blueberry selenium enrichment, is characterized in that, comprises the following steps: After the collected blueberry mycorrhizae are disinfected, they are placed on PDA solid medium containing antibiotics at pH 6-8, cultivated in a constant temperature incubator at a temperature of 25-30°C, and the isolated mycorrhizal fungi are inoculated into the antibiotic-containing medium. Purify and cultivate on PDA medium, inoculate the purified strain on PDA slant, and store at 3-4°C for later use; (2) Reattach the mycorrhizal fungi obtained in step (1) to blueberry seedlings and plants, detect the content of nutrient elements in blueberry leaves and fruits by quantum resonance and ICP-MS, and select mycorrhizal fungi that promote selenium enrichment in blueberries, That's it. 2. a kind of identification method of the mycorrhizal fungus that promotes blueberry selenium as claimed in claim 1, is characterized in that, The screened mycorrhizal fungi were identified by molecular biology and morphological methods to determine the species of mycorrhizal fungi. 3. the identification method of the mycorrhizal fungus that promotes blueberry selenium-enrichment according to claim 2, is characterized in that, The molecular biology identification method is as follows: the genome of the screened mycorrhizal fungi is extracted, and the ITS sequence of the fungal rDNA is used as a universal primer for PCR amplification. The sequences obtained by sequencing are compared and analyzed by BLAST in the GenBank database. In the results, sequences with more than 97% homology were analyzed using CLUSTAL-W software for sequence matching, and the Neighbor-joining method in MEGA6 software was used to construct a phylogenetic tree to determine the phylogenetic status of mycorrhizal fungi. 4. the identification method of the mycorrhizal fungus that promotes blueberry selenium-enrichment according to claim 2, is characterized in that, The mycorrhizal fungus beneficial to blueberry selenium enrichment was determined to be Mucor circinelloides of Mucor circinelloides. 5. a kind of application of the mycorrhizal fungus that promotes blueberry selenium as claimed in claim 1, is characterized in that, It can be used to prepare mycorrhizal fungus preparations and be applied to the production of blueberries.