CN113736671B

CN113736671B - Endophytic fungi AM8.2 for promoting potassium element absorption of camellia oleifera

Info

Publication number: CN113736671B
Application number: CN202111222600.9A
Authority: CN
Inventors: 王晓艳; 吴轩毅; 王晓伟; 苏军
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-10-20
Filing date: 2021-10-20
Publication date: 2023-07-28
Anticipated expiration: 2041-10-20
Also published as: CN113736671A

Abstract

The invention provides endophytic fungi AM8.2 which are obtained from the root screening of woody plant camellia oleifera and promote potassium element absorption of camellia oleifera, and belongs to the technical field of microorganisms. The classification of the endophytic fungi AM8.2 is named as acanthopanax crazyweed @Colletotrichum spinosum.) AM8.2 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.23245 in the year 09 and 01 of 2021. The bacterial fertilizer prepared from endophytic fungi AM8.2 can reduce the use of chemical fertilizers, improve the microbial activity of soil, influence the physicochemical properties of the soil of the camellia oleifera, influence the absorption of endogenous hormones and potassium and nitrogen elements of the camellia oleifera, and promote the growth and development of plants.

Description

Endophytic fungi AM8.2 for promoting potassium element absorption of camellia oleifera

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to endophytic fungi AM8.2 for promoting potassium element absorption of camellia oleifera.

Background

Potassium is an element necessary for the growth and development of crops, and the potassium content in plants is generally 0.2-4.1% of the dry matter mass and is only less than nitrogen. It can regulate plant metabolism, promote carbohydrate synthesis and nitrogen metabolism, raise crop yield, improve fruit quality and strengthen photosynthesis of plant leaf. It can also promote the transportation of photosynthetic products to tubers and improve the synthesis capacity of sucrose in leaves. The potassium can enhance the stress resistance of crops and promote the formation of good quality of crops.

Tea-oil camelliaCamellia oleifera Abel.) Belongs to the genus Camellia of the family Theaceae, and is known as the world four woody oilseed tree species with oil palm, olive and coconut. The oil tea is an important woody edible oil crop in the southern hilly areas of China, has more than 1160 billions of annual output values, and plays a very important role in the forestry industry and the economic development of hilly mountainous areas. Meanwhile, the tea-oil camellia leaves can adsorb harmful substances in the air, and have a certain ecological value. From the actual planting condition of the camellia oleifera in China, most of the camellia oleifera in China has low yield, and the production enthusiasm of farmers is greatly hit. The low yield and low yield are the main problems facing the oil tea industry at present, and the health and sustainable development of the oil tea industry can be promoted only by improving the yield of the oil tea. Mineral nutrient elements have remarkable influence on the growth and yield of the oil tea, and nearly one fourth of the land in China lacks potassium, so that operators have insufficient importance on potash fertilizers, and potassium becomes a main factor influencing the growth and quality of crops, so that the growth and yield increase of the oil tea are promoted, and the potassium absorption rate of the oil tea is improved.

Various endophytic fungi with growth promotion function are screened from plant bodies by students at home and abroad, but the current research is mainly focused on screening related strains of gramineae plants, and researches on woody plants, especially screening and identifying the endophytic fungi of camellia oleifera, are reported. Screening endophytic fungi beneficial to the growth and stress resistance of the camellia oleifera from camellia oleifera roots, and establishing a camellia oleifera-endophytic fungi symbiotic system so as to obtain the endophytic fungi capable of promoting the growth of the camellia oleifera and improving potassium absorption of the camellia oleifera, thereby providing a data basis and a reference basis for enriching strain information of the endophytic fungi of woody plants and manufacturing biological bacterial manure for camellia oleifera forest management.

Disclosure of Invention

The invention aims to provide endophytic fungi AM8.2 for promoting potassium element absorption of tea-oil trees aiming at the problem that the tea-oil trees lack a tea-oil tree-endophytic fungi symbiotic system.

In order to achieve the above purpose, the invention adopts the following technical scheme:

endophytic fungus AM8.2 for promoting potassium element absorption of camellia oleifera, wherein the classification of the endophytic fungus AM8.2 is named as acanthopanax sessiliflorusColletotrichum spinosum.) AM8.2 is preserved in China general microbiological culture Collection center (CGMCC) in the year 09 and 01 of 2021, and has a preservation number of CGMCC No.23245 and a preservation address of North Chen Xilu No.1 and 3 of the Chaoyang area of Beijing city.

The endophytic fungus AM8.2 for promoting potassium element absorption of the camellia oleifera is applied to promoting the growth of the camellia oleifera.

The endophytic fungi AM8.2 is obtained by separating and purifying from the roots of the camellia oleifera, can be prepared into bacterial liquid, and can be prepared into bacterial liquid to promote the growth of camellia oleifera seedlings by a mode of mixing with an organic fertilizer and ditch application.

A bacterial fertilizer containing endophytic fungi AM8.2 bacterial liquid comprises the following components: 1 liter of endophytic fungi AM8.2 bacterial liquid and organic fertilizer: mixing the materials according to the proportion of 10 kg. The preparation method of the endophytic fungi AM8.2 bacterial liquid comprises the following steps: inoculating endophytic fungus AM8.2 into liquid culture medium, culturing at 28deg.C and 220r/min shaking table at constant temperature for 72 hr, diluting the obtained culture solution with sterile water to spore concentration of 0.6X10 ⁹ CFU/ml. The formula of the liquid culture medium is as follows: 20.0g of peptone, 10.0g of yeast extract, 20.0g of glucose, 0.03g of adenine sulfate, 1000ml of ultrapure water and pH 5.6+/-0.2.

The bacterial fertilizer containing the endophytic fungi AM8.2 bacterial liquid is applied to the planting of oil tea.

The invention has the advantages that:

the endophytic fungi AM8.2 is obtained from the root of the woody plant camellia oleifera in a screening way, and the strain can promote the camellia oleifera to absorb potassium elements and nitrogen elements, so that the plant growth can be promoted. The bacterial fertilizer prepared from endophytic fungi AM8.2 can reduce the use of chemical fertilizers, improve the microbial activity of soil, and influence the physicochemical properties of the soil of the camellia oleifera, thereby influencing the absorption of endogenous hormones and potassium and nitrogen elements of the camellia oleifera. Thereby promoting the growth and development of plants.

Description of the drawings:

FIG. 1 is a colony morphology of endophytic fungi AM8.2. Left: a front face; right: and a back surface.

Detailed Description

In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.

Example 1 isolation of tea-oil camellia root endophytic fungi

Culture medium:

(1) Liquid medium YPDA: peptone 20.0g, yeast extract 10.0g, glucose (C) ₆ H ₁₂ O ₆ •H ₂ O) 20.0g, adenine sulfate 0.03g, ultrapure water 1000ml, pH 5.6.+ -. 0.2.

(2) PDA medium (potato dextrose agar medium): 6.0g of potato soaked powder, 20.0g of agar g, 20.0g of glucose and pH 5.6+/-0.2.

Isolation of endophytic fungi

(1) The tissue separation method is adopted, roots of the camellia oleifera are washed clean by running water and then are dried in an ultra-clean bench for tissue surface disinfection, and the operation flow is as follows: 75vol% of absolute ethyl alcohol sterilization, sterile water cleaning for 2-3 times, 15vol% of sodium hypochlorite sterilization and sterile water cleaning for 2-3 times. Cutting phloem of the sterilized root with a sterile blade, cutting into 2mm×2mm size, placing on PDA culture medium, and culturing at 28deg.C under constant temperature and in dark place.

(2) Verification of disinfection effect: and (3) coating sterile water for cleaning the sample in the last step of sterilization on an unused PDA culture medium, and culturing for 4-7 d at a constant temperature of 28 ℃, wherein if the sterile body grows out, the sterilization is clean. And (3) adopting a tissue blotting method, lightly rolling the sterilized sample tissue on an unused PDA culture medium or placing the sample tissue close to the culture medium for 5min, taking the sample tissue away to serve as a reference, and culturing the sample tissue at the constant temperature of 28 ℃ for 4-7 d, wherein the sample tissue is sterilized if aseptic bodies grow out. Each control was repeated 3 times.

Purification of endophytic fungi

After the tissue material is cultured for 3-5 d, mycelia with good colony growth around the tissue are picked by an inoculating needle, strain purification is carried out on the mycelia on a PDA culture medium by a streaking method, the mycelia are inverted in a constant temperature incubator, and the mycelia are cultured for 4-7 d at a constant temperature and in a dark place at 28 ℃. And repeatedly purifying for 3-4 times to obtain a purified strain AM8.2. And (5) inoculating the purified strain into PDA slant culture medium, and preserving at 4 ℃.

3. Morphological characteristics of endophytic fungi AM 8.2:

4 DNA identification of endophytic fungi

Amplifying the 18S rDNA gene sequence (SEQ ID NO. 1) of the endophytic fungus AM8.2 by using a fungus 18S rDNA universal forward and reverse primer (ITS 1: TCCGTAGGTGAACCTGCGG, ITS4: TCCTCCGCTTATTGATATGC); the result of comparing the detected 18S rDNA sequence with the sequence in GenBank database shows that the AM8.2 strain and the acanthopanax strain are @ theColletotrichum spinosum.) On the same branch; combining colony morphology, physiological and biochemical characteristics and 18S rDNA sequence analysis, and preliminarily identifying the bacterial strain as acanthopanaxColletotrichum spinosum.）。

>AM8.2 SEQ ID NO.1：

TCTTCCGTAAGGGTGACCTGCGGAGGGATCATTACTGAGTTTACGCTCTATAACCCTTTGTGAACATACCTAACCGTTGCTTCGGCGGGCGGGAGGTCCGCCTCCCCCCCGGCCCCGCTCGCGGGGCGCCCGCCGGAGGAAAAACCCAACTCTTATTTTAACGACGTCTCTTCTGAGTGGCACAAGCAAATAATCAAAACTTTTAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGCATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCACCGCTTGGCGTTGGGGCTTCCACGGCTGACGTGGGCCCTCAAAGACAGTGGCGGACCCTCGCGGAGCCTCCTTTGCGTAGTAACATACCACCTCGCACCGGGACCCGCAGGGCACTCCTGCCGTAAAACCCCCCAATTTTTACAAGGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCTAAAGCCGGAAGAAAAAATAAC

Example 2

Preparing bacterial liquid: inoculating activated endophytic fungus AM8.2 into 20mL potato glucose liquid culture medium, culturing on constant temperature shaking table for 72 hr (28deg.C, 220 r/min), and sucking 1mLAdding into 2 500mLYPDA culture media, counting spores with blood cell counting plate, diluting the cultured bacterial liquid with sterile water to spore concentration of 0.6X10 ⁹ CFU/ml。

Bacterial fertilizer preparation: the mixed solid bacterial manure comprises the following components: endophytic fungi AM8.2 bacterial liquid: organic fertilizer = 1 liter: 10 kg. The organic fertilizer is prepared by fermenting tobacco foam, rice bran, cow dung, peanut residue, plant ash, humic acid, sheep manure and the like, wherein the mass fraction of organic matters is more than 46%, and the mass fraction of the organic matters is N+P ₂ O ₅ +K ₂ The mass fraction of O is more than 5%.

Experimental protocol: the method adopts an outdoor test, and the camellia oleifera forest land selected by the test is provided by Qingli county Qingli national forest land of Sanming City of Fujian province. And selecting oil tea forests with good growth state as sampling areas in the bacterial manure experiment area and 1 blank control area. The prepared solid bacterial manure is applied to a bacterial manure test area in a watering mode, and the prepared solid bacterial manure is applied to a position 40 centimeters below a projection area of a camellia oleifera forest land in a ditch for 1 month, and soil samples and She Yang are collected at the bottom of 6 months because 7 months are rainy seasons. Three sampling points are randomly selected to sample leaves, roots and soil, and 5 samples are taken from each sampling point to be mixed into 1 sample to be tested.

Influence of bacterial manure on physical and chemical properties of soil

Three physicochemical properties of the soil sample, such as water content, conductivity and pH value, were measured, and the results are shown in Table 1:

TABLE 1 soil moisture content, conductivity and pH

Different lower case letters indicate that the difference is significant at the same treatment level (ttest,p < 0.05）

as can be seen from Table 1, the bacterial manure prepared from the AM8.2 strain has significantly different water content, conductivity and pH value from the control group compared with the control group.

Influence of bacterial manure on oil tea She Nayuan hormone

The concentrations of the standard in the Plant GA ELISA KIT (ml 072782, MLBIO, shanggai), plant IAA ELISA KIT (ml 022829, MLBIO, shanggai), plant ABA ELISA KIT (ml 064270, MLBIO, shanggai) and Plant BR ELISA KIT (ml 036309, MLBIO, shanggai) kits were plotted on the abscissa, the measured OD values on the ordinate, and a standard curve was plotted to obtain a linear regression equation, and the sample content was calculated (Table 2).

Table 2 GA, IAA, ABA, BR content

as shown in Table 2, the bacterial manure prepared from the AM8.2 strain had a GA (pmol/ml) content of 211.30.+ -. 11.92 and an ABA (ng/ml) content of 140.26.+ -. 4.51, which were significantly different from those of the control group.

Influence of bacterial manure on absorption of nitrogen, phosphorus and potassium elements by camellia oleifera

The relative contents of three elements, such as N, P, K, endogenous to the tea-oil tree leaves were measured, and the results are shown in table 3:

table 3 relative content of endogenous N, P, K and other three elements

as can be obtained from table 3, compared with the control treatment group, the bacterial manure prepared from the AM8.2 strain has a relative content of K of 1.39±0.10, which is significantly different from the control group, indicating that the strain AM8.2 can significantly promote the absorption of potassium (K) by oil tea; meanwhile, the relative content of N is 1.56+/-0.21, and compared with a control group, the N is also obviously improved.

Soil is a necessary condition for plant growth and development and nutrient substance drawing, the physical and chemical properties of the soil are comprehensive reflection of climate, vegetation, topography and soil structure, and the application of microbial fertilizer can reduce the use of chemical fertilizer and improve the microbial activity of the soil, thereby influencing the growth and development of plants. The experiment can obtain that the organic bacterial fertilizer number prepared from endophytic fungi AM8.2 has positive influence on the soil growth environment of the camellia oleifera, and can influence the physical and chemical properties of the soil of the camellia oleifera to a certain extent, thereby influencing the absorption of endogenous hormones and potassium of the camellia oleifera.

The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

SEQUENCE LISTING

<110> Wang Xiaoyan, wu Xuanyi, wang Xiaowei

<120> endophytic fungus AM8.2 for promoting potassium absorption of camellia oleifera

<130> 3

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 581

<212> DNA

<213> SEQ ID NO.1

<400> 1

tcttccgtaa gggtgacctg cggagggatc attactgagt ttacgctcta taaccctttg 60

tgaacatacc taaccgttgc ttcggcgggc gggaggtccg cctccccccc ggccccgctc 120

gcggggcgcc cgccggagga aaaacccaac tcttatttta acgacgtctc ttctgagtgg 180

cacaagcaaa taatcaaaac ttttaacaac ggatctcttg gttctggcat cgatgaagaa 240

cgcagcgaaa tgcgataagt aatgtgaatt gcagaattca gtgaatcatc gaatctttga 300

acgcacattg cgcccgccag cattctggcg ggcatgcctg ttcgagcgtc atttcaaccc 360

tcaagcaccg cttggcgttg gggcttccac ggctgacgtg ggccctcaaa gacagtggcg 420

gaccctcgcg gagcctcctt tgcgtagtaa cataccacct cgcaccggga cccgcagggc 480

actcctgccg taaaaccccc caatttttac aaggttgacc tcggatcagg taggaatacc 540

cgctgaactt aagcatatct aaagccggaa gaaaaaataa c 581

<210> 2

<211> 19

<212> DNA

<213> ITS1

<400> 2

tccgtaggtg aacctgcgg 19

<210> 3

<211> 20

<212> DNA

<213> ITS4

<400> 3

tcctccgctt attgatatgc 20

Claims

1. Endophytic fungi AM8.2 for promoting potassium element absorption of camellia oleifera is characterized in that: the classification of the endophytic fungi AM8.2 is named as acanthopanax crazyweed @Colletotrichum spinosum.) AM8.2 is preserved in China general microbiological culture Collection center (CGMCC) in the year 09 and 01 of 2021, and has a preservation number of CGMCC No.23245 and a preservation address of North Chen Xilu No.1 and 3 of the Chaoyang area of Beijing city.

2. The use of endophytic fungus AM8.2 for promoting potassium element absorption of camellia oleifera in promoting growth of camellia oleifera as claimed in claim 1.

3. A bacterial fertilizer containing endophytic fungi AM8.2 bacterial liquid is characterized in that: the preparation of the bacterial fertilizer comprises the following steps: 1 liter of endophytic fungi AM8.2 bacterial liquid and organic fertilizer: mixing the materials in a proportion of 10 kg to obtain a solid bacterial fertilizer; the preparation method of the endophytic fungi AM8.2 bacterial liquid comprises the following steps: inoculating endophytic fungus AM8.2 of claim 1 into liquid culture medium, culturing at 28deg.C and 220r/min shaking table constant temperature for 72 hr, diluting the obtained culture solution with sterile water to spore concentration of 0.6X10 ⁹ CFU/ml。

4. A bacterial manure according to claim 3, wherein: the formula of the liquid culture medium is as follows: 20.0g of peptone, 10.0g of yeast extract, 20.0g of glucose, 0.03g of adenine sulfate, 1000ml of ultrapure water and pH 5.6+/-0.2.

5. Use of the bacterial manure of claim 3 in camellia oleifera planting.