CN111184031A - Liquid composite mycorrhizal inoculant for promoting growth of blueberry seedlings and preparation method thereof - Google Patents
Liquid composite mycorrhizal inoculant for promoting growth of blueberry seedlings and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a liquid composite mycorrhizal inoculant for promoting growth of blueberry seedlings and a preparation method thereof, wherein the liquid composite mycorrhizal inoculant comprises the following steps: (1) carrying out propagation culture on a single strain; (2) preparing a complex microbial inoculum; the strain of the mycorrhizal fungi agent is 3 mycorrhizal fungi T1, T2 and T3 which are separated from the root system of Rhododendron (Rhododendron simsii); the beneficial conditions of the invention are as follows: 1. after the microbial inoculum is applied, the T1+ T2+ T3 liquid compound microbial inoculum has the most remarkable growth promoting effect on the blueberry seedlings; 2. the preparation method saves the operation time and the required materials, reduces the discharge of waste materials, improves the operation efficiency in production practice and controls the batch production cost.
Description
Technical Field
The invention relates to a liquid compound mycorrhizal inoculant for promoting growth of blueberry seedlings and a preparation method thereof.
Background
Blueberry (Vaccinium myrobosum) belongs to Vaccinium myrtillus (Vaccinium myrtillus) of Ericaceae, has fine and smooth pulp, is sour and sweet, is rich in anthocyanin and other nutrient components, and is a globally famous economic berry plant. In recent years, the cultivation and production of blueberries in China are rapidly developed, and the blueberries are planted in large scale in nearly 30 provinces, direct prefectures and autonomous regions in China as of 2017. How to promote the growth of the blueberries so as to improve the economic benefit is a fundamental problem in the large-scale planting of the blueberries. Although applying chemical fertilizer or organic fertilizer to the blueberries can help the growth of the blueberries, the chemical fertilizer easily causes soil degradation pollution and hinders organic and green food certification of planting products, and the organic fertilizer is large in volume and weight and inconvenient to transport and apply. The microbial fertilizer takes a microbial living body as a core, and has outstanding advantages in the aspects of maintaining and improving soil fertility, promoting the conversion and absorption of nutrient elements, promoting the growth of crops, antagonizing soil diseases and insect pests, purifying and maintaining ecological system balance and the like. The proper strain type is the key of the microbial fertilizer, and the compound microbial inoculum with the synergistic effect of a plurality of strains is generally more powerful and stable than the fertilizer effect of a single microbial inoculum. The production and preparation of the microbial fertilizer relate to the selection of various parameters such as a culture mode, a culture medium, a container, temperature, time and the like.
Mycorrhiza is a mutual-profit symbiont formed by a higher plant nutrient root system and mycorrhizal fungi hypha, and is a very common natural phenomenon. The roots of plants of the Ericaceae family, except for the Rhododendron berries (Arbutus) and Arctophyllos, are composed of a large number of fine fibrous roots and have no root-hair structure, so that the nutrient absorption capacity is very limited, especially depending on the action of mycorrhiza. Some mycorrhizal fungi have a "broad spectrum," i.e., can form symbiotic relationships with the roots of various ericaceae plants. Ericaceae plants in the same region also often possess similar mycorrhizal fungal species and their composition. However, in practical production of blueberries, on one hand, most blueberry seedlings are aseptic seedlings obtained by tissue culture, and on the other hand, planting soil or culture medium is often disinfected for preventing diseases and insect pests, so that the roots of the blueberries lack necessary symbiotic mycorrhizal fungi, and the growth of the blueberries is influenced to a great extent.
In order to make up for the defects of chemical fertilizers and organic fertilizers, aiming at the problems that blueberries are mycorrhiza-dependent plants and symbiotic mycorrhiza is lacked under cultivation conditions, a compound mycorrhiza microbial inoculum is needed to improve the growth of the blueberries.
Disclosure of Invention
In order to solve the problems, the invention aims to provide the liquid compound mycorrhizal fungi agent for promoting the growth of the blueberry seedlings, which promotes the rooting and growth of the blueberry seedlings, has short preparation method time and less waste discharge, and the preparation method thereof.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a liquid compound mycorrhizal inoculant for promoting growth of blueberry seedlings and a preparation method thereof comprise the following steps:
(1) and (3) propagation culture of single strains:
activating strains: respectively selecting a small amount of hyphae from the preserved strains T1, T2 and T3 in an ultraclean workbench, inoculating the hyphae to a potato glucose agar culture medium plate, placing the plate in the dark at the temperature of 25 ℃ for culturing for 2 to 3 days, and then inoculating the hyphae to a new potato glucose agar culture medium plate again for culturing for 2 to 3 days;
preparing a shaking table fermentation culture medium: cleaning and peeling potatoes, cutting the potatoes into small pieces, weighing 80-120 g of the potatoes, adding 700-800 ml of distilled water, pouring the obtained mixture into a stirrer, fully crushing the obtained mixture for 2-3 min to obtain slurry, adding 20 g of glucose and 0.1 g of penicillin, fully dissolving the obtained product, finally setting the volume of the distilled water to 1000 ml, subpackaging the obtained product into 250 ml triangular conical flasks, pouring 150 ml of the obtained product into each flask, carrying out high-pressure sterilization at 121 ℃ for 20 min, and cooling the obtained product for later use;
fermenting by a shaking table: cutting 5 mm multiplied by 5 mm of mycelium blocks from edges of activated cultured T1, T2 and T3 bacterial colonies in an ultra-clean workbench, adding 3 mycelium blocks into a shaking table fermentation medium per bottle, and carrying out shaking table fermentation for 10-14 days at 25 ℃ at 120-200 r/min until the upper liquid of the culture is clear;
(2) preparing a complex microbial inoculum:
preparing a single-strain hypha suspension: respectively pouring the culture obtained by propagation culture of three single strains of T1, T2 and T3 into a clean stirrer, crushing for 30 s, and adding sodium alginate at a ratio of 3 g/100 ml to obtain mycelium suspension;
preparing a complex microbial inoculum: mixing single strain hypha suspension prepared from T1, T2 and T3 in equal proportion to obtain 4 liquid composite mycorrhizal inoculants combined by T1+ T2, T1+ T3, T2+ T3, T1+ T2+ T3;
application of the microbial inoculum: diluting the prepared composite microbial inoculum by 50-100 times with clear water, applying the diluted composite microbial inoculum to rhizosphere soil of blueberry seedlings, and counting growth data of the blueberries 2 months after the bacterial application;
the strain of the mycorrhizal fungi agent is 3 mycorrhizal fungi T1, T2 and T3 which are separated from a root system of Rhododendron (Rhododendron simsii) and have the following sequences:
the mycorrhizal fungi T1 was identified as a homologous species of Phialocephala fortinii, and the Internal Transcribed Spacer (ITS) sequence of ribosomal deoxyribonucleic acid (rDNA) is as follows:
CCGTTGGAACAGCGGTTAGGAGCTTACACCCACCCGTGTTTACATACTATTGTTGCTTTGGCGGGCCGTGGCCTCCACTGCGGGCTCTGCTCGTGTGTGCCCGCCAGAGAACCAAACTCTGAATGTTAGTGATGTCTGAGTACTATCTAATAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTGTGGTATTCCGCAGGGCATGCCTGTTCGAGCGTCATTTAACCACTCACGCCCGGCGTGGTATTGGGGCACGCGGTCTCCGCGGCCCTCAAAATTAGTGGCGGTGCCGGTGGGCTCTAAGCGTAGTACATACTCCCGCTATAGAGTTCCCCCGGTGGCTCGCCAGAACCCCCAATTTTTTTACAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA；
mycorrhizal fungi T2 was identified as a homologous species of Ilyonectria leucospermi, and ITS ribosomal deoxyribonucleic acid (rDNA) Internal Transcribed Spacer (ITS) sequence was as follows:
CACCCCTGTGACATACCATTTGTTGCCTCGGCGGTGCCTGCTTCGGCAGCCCGCCAGAGGACCCAAACCCTTGATTTTATACAGTATCTTCTGAGTAAATGATTAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCCCCCGGGCTTGGTGTTGGAGATCGGCGTGCCCTCCGGGGCGCGCCGGCTCCCAAATATAGTGGCGGTCTCGCTGTAGCTTCCTCTGCGTAGTAGCACACCTCGCACTGGGAAACAGCGTGGCCACGCCGTTAAACCCCCCACTTCTGAAAGGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA；
mycorrhizal fungi T3 was identified as a homolog of Nemania sp, and ITS ribosomal deoxyribonucleic acid (rDNA) Internal Transcribed Spacer (ITS) sequence was as follows:
TCAACCCATGTGAACATACCTCATGTTGCCTCGGCAGGTCGTGCCTCCCTCGTAGGTCCTACCCTGTAGGCTCCTACCCGGAAGGCGCGGGTACCCCTGCCGGTGGCCCAGGAAACTCTGTCTCATCGTTGAATTCTGAACCTATAACTAAATAAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCATTAGTATTCTAGTGGGCATGCCTGTTCGAGCGTCATTTCAACCCTTAAGCCCTTGTTGCTTAGCGTTGGGAGCCTACGGCACCGTAGCTCCCCAAAGTCAGTGGCGGAGCCGGCTCACACTCTAGACGTAGTAATTTCTCACCTCGCCTATAGTTGGACCGGTCCCCTGCCGTAAAACGCCCCAGTATTTAAAAGGTTGACCTCGAATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGGCGGAGGAA。
preferably, the optimal cultivation time of the shaking table fermentation of the strains T1, T2 and T3 in the step (1) is 14 days, 12 days and 10 days respectively.
Preferably, the method for applying the microbial inoculum in the step (2) is to annularly inject the compound microbial inoculum into the rhizosphere soil of the blueberry seedlings by 2 cm around the root systems of the blueberry seedlings, and the compound microbial inoculum is applied once every 10 days and is continuously applied for 3 times.
Preferably, after the microbial inoculum is applied in the step (2), the T1+ T2+ T3 liquid compound microbial inoculum has the most remarkable growth promotion effect on blueberry seedlings, the height of the liquid compound microbial inoculum is 11.86 cm, the weight of the upper fresh weight is increased by 1.08 g, the weight of the lower fresh weight is increased by 0.28g, the weight of the upper dry weight is increased by 0.38 g, and the weight of the lower dry weight is increased by 0.09 g.
The beneficial conditions of the invention are as follows:
1. after the microbial inoculum is applied, the T1+ T2+ T3 liquid composite microbial inoculum has the most remarkable growth promotion effect on blueberry seedlings, the height is increased to 11.86 cm, the weight of the upper fresh weight is increased to 1.08 g, the weight of the lower fresh weight is increased to 0.28g, the weight of the upper dry weight is increased to 0.38 g, and the weight of the lower dry weight is increased to 0.09 g;
2. compared with the conventional preparation method of the potato glucose culture medium, the preparation method of the invention replaces the operations of boiling, filtering and the like by crushing the potato glucose culture medium into the slurry by the stirrer, thereby saving the operation time and the required materials, reducing the discharge of waste materials, improving the operation efficiency in the production practice and controlling the batch production cost.
Detailed Description
A liquid compound mycorrhizal inoculant for promoting growth of blueberry seedlings and a preparation method thereof comprise the following steps:
(1) and (3) propagation culture of single strains:
activating strains: respectively selecting a small amount of hyphae from the preserved strains T1, T2 and T3 in an ultraclean workbench, inoculating the hyphae to a potato glucose agar culture medium plate, placing the plate in the dark at the temperature of 25 ℃ for culturing for 2 to 3 days, and then inoculating the hyphae to a new potato glucose agar culture medium plate again for culturing for 2 to 3 days;
preparing a shaking table fermentation culture medium: cleaning and peeling potatoes, cutting the potatoes into small pieces, weighing 80-120 g of the potatoes, adding 700-800 ml of distilled water, pouring the obtained mixture into a stirrer, fully crushing the obtained mixture for 2-3 min to obtain slurry, adding 20 g of glucose and 0.1 g of penicillin, fully dissolving the obtained product, finally setting the volume of the distilled water to 1000 ml, subpackaging the obtained product into 250 ml triangular conical flasks, pouring 150 ml of the obtained product into each flask, carrying out high-pressure sterilization at 121 ℃ for 20 min, and cooling the obtained product for later use;
fermenting by a shaking table: cutting 5 mm multiplied by 5 mm of mycelium blocks from the edges of activated cultured T1, T2 and T3 bacterial colonies in an ultra-clean workbench, adding 3 mycelium blocks into a shaking table fermentation medium per bottle, and carrying out shaking table fermentation for 10-14 days at 25 ℃ and 150r/min until the upper liquid of the culture is clear;
(2) preparing a complex microbial inoculum:
preparing a single-strain hypha suspension: respectively pouring the culture obtained by propagation culture of three single strains of T1, T2 and T3 into a clean stirrer, crushing for 30 s, and adding sodium alginate at a ratio of 3 g/100 ml to obtain mycelium suspension;
preparing a complex microbial inoculum: mixing single strain hypha suspension prepared from T1, T2 and T3 in equal proportion to obtain 4 liquid composite mycorrhizal inoculants combined by T1+ T2, T1+ T3, T2+ T3, T1+ T2+ T3;
application of the microbial inoculum: diluting the prepared composite microbial inoculum by 100 times with clear water, applying the diluted composite microbial inoculum to rhizosphere soil of blueberry seedlings, and counting growth data of the blueberries 2 months after the bacterial application;
the strain of the mycorrhizal fungi agent is 3 mycorrhizal fungi T1, T2 and T3 which are separated from a root system of Rhododendron (Rhododendron simsii) and have the following sequences:
the mycorrhizal fungi T1 was identified as a homologous species of Phialocephala fortinii, and the Internal Transcribed Spacer (ITS) sequence of ribosomal deoxyribonucleic acid (rDNA) is as follows:
CCGTTGGAACAGCGGTTAGGAGCTTACACCCACCCGTGTTTACATACTATTGTTGCTTTGGCGGGCCGTGGCCTCCACTGCGGGCTCTGCTCGTGTGTGCCCGCCAGAGAACCAAACTCTGAATGTTAGTGATGTCTGAGTACTATCTAATAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTGTGGTATTCCGCAGGGCATGCCTGTTCGAGCGTCATTTAACCACTCACGCCCGGCGTGGTATTGGGGCACGCGGTCTCCGCGGCCCTCAAAATTAGTGGCGGTGCCGGTGGGCTCTAAGCGTAGTACATACTCCCGCTATAGAGTTCCCCCGGTGGCTCGCCAGAACCCCCAATTTTTTTACAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA；
mycorrhizal fungi T2 was identified as a homologous species of Ilyonectria leucospermi, and ITS ribosomal deoxyribonucleic acid (rDNA) Internal Transcribed Spacer (ITS) sequence was as follows:
CACCCCTGTGACATACCATTTGTTGCCTCGGCGGTGCCTGCTTCGGCAGCCCGCCAGAGGACCCAAACCCTTGATTTTATACAGTATCTTCTGAGTAAATGATTAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCCCCCGGGCTTGGTGTTGGAGATCGGCGTGCCCTCCGGGGCGCGCCGGCTCCCAAATATAGTGGCGGTCTCGCTGTAGCTTCCTCTGCGTAGTAGCACACCTCGCACTGGGAAACAGCGTGGCCACGCCGTTAAACCCCCCACTTCTGAAAGGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA；
mycorrhizal fungi T3 was identified as a homolog of Nemania sp, and ITS ribosomal deoxyribonucleic acid (rDNA) Internal Transcribed Spacer (ITS) sequence was as follows:
TCAACCCATGTGAACATACCTCATGTTGCCTCGGCAGGTCGTGCCTCCCTCGTAGGTCCTACCCTGTAGGCTCCTACCCGGAAGGCGCGGGTACCCCTGCCGGTGGCCCAGGAAACTCTGTCTCATCGTTGAATTCTGAACCTATAACTAAATAAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCATTAGTATTCTAGTGGGCATGCCTGTTCGAGCGTCATTTCAACCCTTAAGCCCTTGTTGCTTAGCGTTGGGAGCCTACGGCACCGTAGCTCCCCAAAGTCAGTGGCGGAGCCGGCTCACACTCTAGACGTAGTAATTTCTCACCTCGCCTATAGTTGGACCGGTCCCCTGCCGTAAAACGCCCCAGTATTTAAAAGGTTGACCTCGAATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGGCGGAGGAA。
by the method, the optimal culture time of shaking table fermentation of strains T1, T2 and T3 is 14 days, 12 days and 10 days respectively.
The dilution times in the application of the microbial inoculum and the application dosage are specifically shown in the following table:
selecting 150 blueberry tissue culture seedlings which pass through seedling hardening and grow more consistently, cleaning the blueberry seedlings in clear water to obtain a root culture medium, transplanting the blueberry seedlings into a hole tray, planting 1 seedling in each hole, and using peat soil and vermiculite as a culture medium according to the ratio of 3: 1, mixing in proportion, performing annular injection on the compound microbial inoculum prepared according to the requirement of the invention in blueberry seedling rhizosphere soil by surrounding 2 cm of blueberry seedling root systems with the upper caliber of 60 mm, the bottom of 28 mm and the depth of 53 mm, performing application once every 10 days, applying 5 ml each time and continuously applying for 3 times, developing a comparison test for influences of different compound microbial inoculum types on growth effects of blueberry seedlings, and performing data statistics 2 months after microbial application.
The statistical results of plant height increase, upper fresh weight increase, lower fresh weight increase, upper dry weight increase and lower dry weight increase of blueberry seedlings by applying different complex microbial agents are shown in the following table. As shown in the table, in the experiment of the influence of different compound microbial inoculums on blueberries, the T1+ T2+ T3 compound microbial inoculums have the most obvious effect on promoting the growth of the blueberries, wherein the plant height is increased to 11.86 cm, the upper fresh weight is increased to 1.08 g, the lower fresh weight is increased to 0.28g, the upper dry weight is increased to 0.38 g, the lower fresh weight is increased to 0.09 g, and all indexes are obviously higher than those of other treatment groups.
| Processing number | Plant height increase (cm) | Fresh weight gain (g) | Weight gain (g) | Increase in Upper Dry weight (g) | Increase in lower Dry weight (g) |
| T1+T2 | 8.77 | 0.56 | 0.13 | 0.20 | 0.05 |
| T2+T3 | 4.92 | 0.23 | 0.05 | 0.08 | 0.02 |
| T1+T3 | 4.54 | 0.31 | 0.07 | 0.11 | 0.02 |
| T1+T2+T3 | 11.86 | 1.08 | 0.28 | 0.38 | 0.09 |
| CK | 3.58 | 0.22 | 0.06 | 0.09 | 0.03 |
It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> administration of natural preservation area of Dachuanshan national level in Zhejiang province
<120> preparation method of liquid composite mycorrhizal inoculant for promoting growth of blueberry seedlings
<130>2018.11.14
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>520
<212>DNA
<213>Rhododendron simsii
<400>1
ccgttggaac agcggttagg agcttacacc cacccgtgtt tacatactat tgttgctttg 60
gcgggccgtg gcctccactg cgggctctgc tcgtgtgtgc ccgccagaga accaaactct 120
gaatgttagt gatgtctgag tactatctaa tagttaaaac tttcaacaac ggatctcttg 180
gttctggcat cgatgaagaa cgcagcgaaa tgcgataagt aatgtgaatt gcagaattca 240
gtgaatcatc gaatctttga acgcacattg cgccctgtgg tattccgcag ggcatgcctg 300
ttcgagcgtc atttaaccac tcacgcccgg cgtggtattg gggcacgcgg tctccgcggc 360
cctcaaaatt agtggcggtg ccggtgggct ctaagcgtag tacatactcc cgctatagag 420
ttcccccggt ggctcgccag aacccccaat ttttttacag gttgacctcg gatcaggtag 480
ggatacccgc tgaacttaag catatcaata agcggaggaa 520
<210>2
<211>495
<212>DNA
<213>Rhododendron simsii
<400>2
cacccctgtg acataccatt tgttgcctcg gcggtgcctg cttcggcagc ccgccagagg 60
acccaaaccc ttgattttat acagtatctt ctgagtaaat gattaaataa atcaaaactt 120
tcaacaacgg atctcttggt tctggcatcg atgaagaacg cagcgaaatg cgataagtaa 180
tgtgaattgc agaattcagt gaatcatcga atctttgaac gcacattgcg cccgccagta 240
ttctggcggg catgcctgtt cgagcgtcat ttcaaccctc aagcccccgg gcttggtgtt 300
ggagatcggc gtgccctccg gggcgcgccg gctcccaaat atagtggcgg tctcgctgta 360
gcttcctctg cgtagtagca cacctcgcac tgggaaacag cgtggccacg ccgttaaacc 420
ccccacttct gaaaggttga cctcggatca ggtaggaata cccgctgaac ttaagcatat 480
caataagcgg aggaa 495
<210>3
<211>535
<212>DNA
<213>Rhododendron simsii
<400>3
tcaacccatg tgaacatacc tcatgttgcc tcggcaggtc gtgcctccct cgtaggtcct 60
accctgtagg ctcctacccg gaaggcgcgg gtacccctgc cggtggccca ggaaactctg 120
tctcatcgtt gaattctgaa cctataacta aataagttaa aactttcaac aacggatctc 180
ttggttctgg catcgatgaa gaacgcagcg aaatgcgata agtaatgtga attgcagaat 240
tcagtgaatc atcgaatctt tgaacgcaca ttgcgcccat tagtattcta gtgggcatgc 300
ctgttcgagc gtcatttcaa cccttaagcc cttgttgctt agcgttggga gcctacggca 360
ccgtagctcc ccaaagtcag tggcggagcc ggctcacact ctagacgtag taatttctca 420
cctcgcctat agttggaccg gtcccctgcc gtaaaacgcc ccagtattta aaaggttgac 480
ctcgaatcag gtaggaatac ccgctgaact taagcatatc aataaggcgg aggaa 535
Claims (4)
1. A liquid compound mycorrhizal inoculant for promoting growth of blueberry seedlings and a preparation method thereof are characterized by comprising the following steps:
(1) and (3) propagation culture of single strains:
activating strains: respectively selecting a small amount of hyphae from the preserved strains T1, T2 and T3 in an ultraclean workbench, inoculating the hyphae to a potato glucose agar culture medium plate, placing the plate in the dark at the temperature of 25 ℃ for culturing for 2 to 3 days, and then inoculating the hyphae to a new potato glucose agar culture medium plate again for culturing for 2 to 3 days;
preparing a shaking table fermentation culture medium: cleaning and peeling potatoes, cutting the potatoes into small pieces, weighing 80-120 g of the potatoes, adding 700-800 ml of distilled water, pouring the obtained mixture into a stirrer, fully crushing the obtained mixture for 2-3 min to obtain slurry, adding 20 g of glucose and 0.1 g of penicillin, fully dissolving the obtained product, finally setting the volume of the distilled water to 1000 ml, subpackaging the obtained product into 250 ml triangular conical flasks, pouring 150 ml of the obtained product into each flask, carrying out high-pressure sterilization at 121 ℃ for 20 min, and cooling the obtained product for later use;
fermenting by a shaking table: cutting 5 mm multiplied by 5 mm of mycelium blocks from edges of activated cultured T1, T2 and T3 bacterial colonies in an ultra-clean workbench, adding 3 mycelium blocks into a shaking table fermentation medium per bottle, and carrying out shaking table fermentation for 10-14 days at 25 ℃ at 120-200 r/min until the upper liquid of the culture is clear;
(2) preparing a complex microbial inoculum:
preparing a single-strain hypha suspension: respectively pouring the culture obtained by propagation culture of three single strains of T1, T2 and T3 into a clean stirrer, crushing for 30 s, and adding sodium alginate at a ratio of 3 g/100 ml to obtain mycelium suspension;
preparing a complex microbial inoculum: mixing single strain hypha suspension prepared from T1, T2 and T3 in equal proportion to obtain 4 liquid composite mycorrhizal inoculants combined by T1+ T2, T1+ T3, T2+ T3, T1+ T2+ T3;
application of the microbial inoculum: diluting the prepared composite microbial inoculum by 50-100 times with clear water, applying the diluted composite microbial inoculum to rhizosphere soil of blueberry seedlings, and counting growth data of the blueberries 2 months after the bacterial application;
the strain of the mycorrhizal fungi agent is 3 mycorrhizal fungi T1, T2 and T3 which are separated from a root system of Rhododendron (Rhododendron simsii) and have the following sequences:
the mycorrhizal fungi T1 was identified as a homologous species of Phialocephala fortinii, and the Internal Transcribed Spacer (ITS) sequence of ribosomal deoxyribonucleic acid (rDNA) is as follows:
CCGTTGGAACAGCGGTTAGGAGCTTACACCCACCCGTGTTTACATACTATTGTTGCTTTGGCGGGCCGTGGCCTCCACTGCGGGCTCTGCTCGTGTGTGCCCGCCAGAGAACCAAACTCTGAATGTTAGTGATGTCTGAGTACTATCTAATAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTGTGGTATTCCGCAGGGCATGCCTGTTCGAGCGTCATTTAACCACTCACGCCCGGCGTGGTATTGGGGCACGCGGTCTCCGCGGCCCTCAAAATTAGTGGCGGTGCCGGTGGGCTCTAAGCGTAGTACATACTCCCGCTATAGAGTTCCCCCGGTGGCTCGCCAGAACCCCCAATTTTTTTACAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA；
mycorrhizal fungi T2 was identified as a homologous species of Ilyonectria leucospermi, and ITS ribosomal deoxyribonucleic acid (rDNA) Internal Transcribed Spacer (ITS) sequence was as follows:
CACCCCTGTGACATACCATTTGTTGCCTCGGCGGTGCCTGCTTCGGCAGCCCGCCAGAGGACCCAAACCCTTGATTTTATACAGTATCTTCTGAGTAAATGATTAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCCCCCGGGCTTGGTGTTGGAGATCGGCGTGCCCTCCGGGGCGCGCCGGCTCCCAAATATAGTGGCGGTCTCGCTGTAGCTTCCTCTGCGTAGTAGCACACCTCGCACTGGGAAACAGCGTGGCCACGCCGTTAAACCCCCCACTTCTGAAAGGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA；
mycorrhizal fungi T3 was identified as a homolog of Nemania sp, and ITS ribosomal deoxyribonucleic acid (rDNA) Internal Transcribed Spacer (ITS) sequence was as follows:
TCAACCCATGTGAACATACCTCATGTTGCCTCGGCAGGTCGTGCCTCCCTCGTAGGTCCTACCCTGTAGGCTCCTACCCGGAAGGCGCGGGTACCCCTGCCGGTGGCCCAGGAAACTCTGTCTCATCGTTGAATTCTGAACCTATAACTAAATAAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCATTAGTATTCTAGTGGGCATGCCTGTTCGAGCGTCATTTCAACCCTTAAGCCCTTGTTGCTTAGCGTTGGGAGCCTACGGCACCGTAGCTCCCCAAAGTCAGTGGCGGAGCCGGCTCACACTCTAGACGTAGTAATTTCTCACCTCGCCTATAGTTGGACCGGTCCCCTGCCGTAAAACGCCCCAGTATTTAAAAGGTTGACCTCGAATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGGCGGAGGAA。
2. the method for preparing a liquid complex mycorrhizal agent for promoting growth of blueberry seedlings according to claim 1, wherein the optimal cultivation time of shake fermentation of strains T1, T2 and T3 in step (1) is 14 days, 12 days and 10 days respectively.
3. The method for preparing the liquid complex mycorrhizal fungi agent for promoting growth of blueberry seedlings as claimed in claim 1, wherein the application method of the fungi agent in step (2) is to inject the complex fungi agent into blueberry seedling rhizosphere soil circularly 2 cm around blueberry seedling root system, and the complex fungi agent is applied once every 10 days and is applied 3 times continuously.
4. The method for preparing the liquid complex mycorrhizal inoculant for promoting growth of blueberry seedlings according to claim 1 or 3, wherein after the inoculant is applied in the step (2), the T1+ T2+ T3 liquid complex inoculant has the most remarkable growth promoting effect on blueberry seedlings, the increase is 11.86 cm, the upper fresh weight is increased by 1.08 g, the lower fresh weight is increased by 0.28g, the upper dry weight is increased by 0.38 g, and the lower dry weight is increased by 0.09 g.
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