CN110305794B - Saline-alkali tolerant trichoderma strain, separation and screening method, culture method, application and use method - Google Patents

Abstract

The invention relates to the technical field of microorganisms, in particular to a saline-alkali tolerant trichoderma strain separated from an extremely arid desert area, a separation and screening method, a culture method, an application and a use method. The strain is Trichoderma harzianum-17, and is preserved in China center for type culture Collection with the preservation number: CCTCC NO: and M2018538. The culture method comprises culturing Trichoderma harzianum-17 in dark at 25-28 deg.C on potato glucose agar medium for at least 72h. Use of a saline-alkali tolerant trichoderma strain for promoting seed germination, seedling growth or/and root growth of a plant. The application method comprises the step of irrigating plants with a bacterial suspension of saline-alkali tolerant trichoderma strains. The invention solves the problem that trichoderma with the functions of salt decomposition and growth promotion in the saline-alkali stress environment does not exist in the prior art, and provides saline-alkali tolerant growth-promoting trichoderma which can enhance the stress resistance of crops and promote the growth of the crops in the saline-alkali environment.

Description

Saline-alkali tolerant trichoderma strain, separation and screening method, culture method, application and use method

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to a saline-alkali tolerant trichoderma strain, a separation and screening method, a culture method, an application and a use method.

Background

The improvement and utilization of saline-alkali soil has been extensively and deeply studied for a long time at home and abroad, and a large amount of manpower and material resources are invested. In the 80 s, foreign countries mostly concentrated on the application of chemical modifiers and the damage of the alkalization layer in the deep ploughing layer. China adopts the methods of changing the alkali of the rice, applying organic fertilizer, planting green manure, leveling land and the like. The treatment of saline-alkali soil can be roughly divided into three categories according to the nature of the improvement: (1) a physical repair method; (2) a chemical repair method; and (3) a bioremediation method.

The physical repairing method belongs to non-biological measures and mainly comprises the steps of draining water, flushing, loosening soil, fertilizing, spreading sand and pressing alkali and the like. The chemical repairing method also belongs to non-biological measures, and mainly comprises the addition of modifiers such as polymer, gypsum, zeolite, furfural residue and the like. The invention discloses a saline-alkali soil conditioner, such as Liuyixian and the like, which is prepared by adding water into polymaleic acid, sodium alkyl benzene sulfonate, zinc sulfate or copper sulfate. The bioremediation of saline-alkali soil mainly comprises phytoremediation and microbial remediation. The plant restoration is mainly based on the planting of salt-tolerant plants. The microbial remediation mainly utilizes saline-alkali resistant microorganisms as microbial agents to be applied to soil to promote plant growth, so that the purpose of improving saline-alkali soil is achieved. The conventional method for desalting the soil by adopting a physical method or a chemical method for treating the saline-alkali soil by people for a long time needs a large amount of capital and fresh water, and is difficult to popularize comprehensively. The structure of the soil can be changed only by biological improvement, so that the physicochemical properties of the soil are essentially improved. Therefore, in recent years, research on the improvement and utilization of saline-alkali soil is gradually changed from engineering measures to biological measures. The microbial method breaks through the traditional method for improving the saline-alkali soil, and improves the vegetation and the physical and chemical properties of the soil of the saline-alkali soil in a short time on the premise of little human input, thereby providing a new idea for exploring a method for repairing the saline-alkali soil and having important significance for recovering the local ecosystem.

Trichoderma (Trichoderma) fungi belong to Ascomycota, hypocrea (asexual stage Trichoderma). It is a ubiquitous and easily separable filamentous fungus, commonly found in soil, and one of the important constitutive communities of soil microorganisms. Studies have found that trichoderma can enhance the ability of host plants to tolerate abiotic stresses and can relieve environmental stresses of strong stresses such as drought, low temperature, salt and alkali, and heavy metal contamination. According to existing researches, under the conditions of various abiotic stresses such as osmotic pressure, cold or hot pressure, salinity and the like, the trichoderma harzianum T22 can improve the germination rate of seeds and reduce the content of osmotic stress or lipid peroxide, so that oxidative damage is relieved; the trichoderma can reduce the influence of salt damage on plants and promote the plants to grow under the condition of salt stress. Under the condition of salt stress, certain trichoderma can induce plants to generate resistance, and the influence of salt damage on the plants is reduced, so that the plants are promoted to grow under the condition of salt stress.

The salinization of soil is an important factor influencing agricultural production and ecological environment, and how to improve the saline-alkali tolerance of crops and promote the biological treatment and comprehensive development of the salinized soil is a major problem of future agriculture. At present, the salinization soil improvement and utilization mainly adopt an ecological restoration method, wherein saline-alkali tolerant microorganisms break through the defects of the traditional method for improving the saline-alkali land, and improve the vegetation and the physical and chemical properties of the soil of the saline-alkali land in a short time. The research on saline-alkali tolerant microorganisms becomes a development trend of saline-alkali soil improvement in the future. However, the screening and research of trichoderma harzianum which has the effects of salt decomposition and growth promotion and can survive in a saline-alkali environment are few, and the invention patent of China 'saline-alkali tolerant trichoderma harzianum and application thereof' (publication No. CN 103451111B, publication No. 2015.07.01) discloses trichoderma harzianum aiming at the prevention and treatment institute of tobacco damping-off and tobacco black shank in the saline-alkali stress environment. However, no report is found on trichoderma having salt-decomposing and growth-promoting effects under saline-alkali stress environments, and no systematic research is available on biocontrol trichoderma in enhancing the salt tolerance of plants.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides a saline-alkali tolerant trichoderma strain, a separation and screening method, a culture method, an application and a use method, solves the problem that no trichoderma with the effects of salt decomposition and growth promotion in a saline-alkali stress environment exists in the prior art, and provides the saline-alkali tolerant trichoderma capable of enhancing the stress resistance of crops and promoting the growth of the crops in the saline-alkali environment.

The purpose of the invention is realized by the following technical scheme:

a saline-alkali tolerant Trichoderma strain, wherein the strain is Trichoderma harzianum-17, deposited in China center for type culture Collection with the following preservation number: CCTCC NO: and M2018538.

Trichoderma harzianum-17 is separated from Umben and desert and belongs to extreme environment microbe.

The trichoderma harzianum-17 has high sporulation quantity and good saline-alkali tolerance, can effectively relieve the inhibition effect of salt stress on the germination of plant seeds, and has the effect of promoting the germination of the seeds; meanwhile, the growth of plant seedlings can be promoted, and the salt stress inhibition effect can be relieved.

Trichoderma strain: the hypha is transparent, the wall is smooth, and the diameter is 1.7-6.4 μm; chlamydospores are spherical, unipartum, and intergrowth in the middle of hyphae. Conidia are very many on PDA culture medium, most of them are spherical, and few of them are elliptical, most of them are 1.74-3.64X 1.54-3.26 μm, and their wall is smooth, and is slightly rough, and its translucence is light yellow-green. Conidiophores are transparent, have smooth walls, are straight or slightly curved, have enlarged bases and become thinner towards the tops.

Further, the trichoderma strains are tolerant to salt stress caused by NaCl concentration less than or equal to 5%; the trichoderma strains are tolerant to alkali stress caused by pH less than or equal to 8.5.

Further preferably, the Trichoderma strain is tolerant to a NaCl concentration of 5% and to salt stress and alkali stress caused by a combination of pH 8.5.

The method for separating and screening the saline-alkali tolerant trichoderma strains comprises the following steps:

step one, weighing 9-11 parts by weight of soil sample, adding the soil sample into a conical flask containing 90 parts by weight of sterile water, placing the conical flask on a shaking table, oscillating for at least 10min to obtain a soil suspension, and diluting the soil suspension to 10 times by a 10-fold dilution method ^-2 、10 ^-3 Respectively dripping 1ml of suspension into a sterilized culture dish, pouring 15ml of potato glucose agar culture medium cooled to 40-45 ℃, uniformly mixing with the suspension, and placing in a thermostat at 25 ℃ for culturing for 5-7 days after solidification;

and step two, picking the trichoderma harzianum hyphae growing obviously on the culture medium by adopting a tip hypha picking method, transferring the trichoderma harzianum hyphae to a potato glucose agar slant culture medium with the pH of 8.5 and the NaCl concentration of 5%, and culturing for 3-7 days at the temperature of 20-25 ℃ to obtain the trichoderma harzianum M-17. The identification method of the saline-alkali tolerant trichoderma strains comprises the steps of extracting DNA of trichoderma harzianum M-17 by adopting a CTAB method, analyzing ITS sequences by adopting primers ITS1 and ITS4, analyzing TEF sequences by adopting primers EF1-728F and EF1-986R, submitting the sequences obtained after sequencing to local Blast of an ISTH website for sequence comparison, and identifying the species; the nucleotide sequence of the ITS1 is shown as SEQ ID No.1, the nucleotide sequence of the ITS4 is shown as SEQ ID No.2, the nucleotide sequence of the ITS is shown as SEQ ID No.3, the nucleotide sequence of the EF1-728F is shown as SEQ ID No.4, the nucleotide sequence of the EF1-986R is shown as SEQ ID No.5, and the nucleotide sequence of the TEF is shown as SEQ ID No. 6.

The method for culturing the saline-alkali tolerant trichoderma strains comprises the following steps of carrying out dark culture on trichoderma harzianum-17 on a potato glucose agar culture medium at the temperature of 25-28 ℃ for at least 72h; the potato glucose agar culture medium comprises the following components in parts by weight: 200 parts of potato, 1000 parts of distilled water, 15-20 parts of glucose and 8-12 parts of agar powder.

Further, after the trichoderma harzianum-17 is cultured on a potato glucose agar culture medium for 24 hours, the front side of the colony is in a white villous shape of aerial hyphae, and the back side of the colony is colorless; after the trichoderma harzianum-17 is cultured on a potato glucose agar culture medium for 48 hours, light green conidia are generated on the front surface of a colony, the hyphae are loose and villous, and the back surface of the colony is light green; after the trichoderma harzianum-17 is cultured on a potato glucose agar culture medium for 72 hours, colonies overgrow the whole culture medium, hypha of a middle colony on the front side is white villous, a dark green conidium is generated on the outer ring, and the back of the colony is green.

The saline-alkali tolerant trichoderma strain is applied to promoting germination of plant seeds, seedling growth or/and root growth.

Further, use of the saline alkali tolerant trichoderma strain to alleviate salt stress inhibition during seed germination, seedling growth, or/and root growth of a plant.

According to experimental results, the trichoderma suspension seed soaking can effectively relieve the inhibition effect of salt stress on alfalfa seed germination, and has the effect of promoting seed germination; the trichoderma suspension can promote the growth of alfalfa seedlings, improve the chlorophyll content, increase photosynthesis and relieve the salt stress inhibition; the trichoderma suspension can effectively relieve the inhibition effect of salt stress on the roots of alfalfa seedlings and promote the growth of the roots.

According to the experimental results, the seed soaking of the trichoderma suspension can improve the rice seed germination and relieve the inhibition effect of salt stress; the trichoderma suspension can promote the growth of rice seedlings, improve the chlorophyll content, increase photosynthesis and relieve the salt stress inhibition; the trichoderma suspension can relieve the inhibition effect of salt stress on the roots of rice seedlings and has a certain promotion effect on the growth of the roots;

under the condition of salt stress, the germination of alfalfa and rice seeds and the growth of seedlings are severely inhibited. By soaking the tested trichoderma suspension, the damage of salt stress to the germination of alfalfa and rice seeds can be effectively reduced, the photosynthesis of the plant chlorophyll is protected by promoting the synthesis of the plant chlorophyll, the normal growth of the plant and the growth of seedlings are maintained, and the chlorophyll content is increased.

Further, the plants are alfalfa and rice.

The method for promoting the growth of plants by using the saline-alkali tolerant trichoderma strains, irrigating the plants by using a bacterial suspension containing the saline-alkali tolerant trichoderma strains according to any one of claims 1 to 9; the using amount of the bacterial suspension for irrigating each plant is 50ml, and the content of the trichoderma strains in the bacterial suspension is (0.9-1.1) multiplied by 10 ⁸ cfu/ml。

Biological material preservation description:

and (3) classification and naming: trichoderma harzianum (Trichoderma harzianum-17);

the preservation organization: china center for type culture Collection;

the preservation organization is abbreviated as: CCTCC;

the preservation number is: CCTCC NO of M2018538;

the preservation date is as follows: 8 and 13 months in 2018.

The beneficial effects of the invention are:

1. the trichoderma harzianum-17 has high sporulation quantity, can promote the growth, can enhance the stress resistance of crops, and can promote the growth of the crops in a saline-alkali environment; the saline-alkali resistance is good, the inhibition effect of salt stress on plant seed germination can be effectively relieved, and the effect of promoting seed germination is achieved; meanwhile, the growth of plant seedlings can be promoted, and the salt stress inhibition effect can be relieved.

2. According to experimental results, the trichoderma suspension seed soaking can effectively relieve the inhibition effect of salt stress on alfalfa seed germination, and has the effect of promoting seed germination; the trichoderma suspension can promote the growth of alfalfa seedlings, improve the chlorophyll content, increase photosynthesis and relieve the salt stress inhibition; the trichoderma suspension can effectively relieve the inhibition effect of salt stress on the roots of the alfalfa seedlings and promote the growth of the roots;

3. according to the experimental results, the seed soaking of the trichoderma suspension can improve the rice seed germination and relieve the inhibition effect of salt stress; the trichoderma suspension can promote the growth of rice seedlings, improve the chlorophyll content, increase photosynthesis and relieve the salt stress inhibition; the trichoderma suspension can relieve the inhibition effect of salt stress on the roots of rice seedlings and has a certain promotion effect on the growth of the roots;

4. under the condition of salt stress, the germination of alfalfa and rice seeds and the growth of seedlings are severely inhibited. By soaking seeds in the tested trichoderma suspension, the harm of salt stress to the germination of alfalfa and rice seeds can be effectively reduced, the photosynthesis of the plant chlorophyll is protected by promoting the synthesis of the plant chlorophyll, the normal growth and seedling growth of the plant are maintained, and the chlorophyll content is increased.

Drawings

FIG. 1 is a comparison of alfalfa seed germination parameters under salt stress for a test Trichoderma strain;

FIG. 2 is a comparison of rice seed germination parameters under salt stress for test Trichoderma strains;

FIG. 3 is a graph of the effect of test Trichoderma strains on alfalfa seedling root systems;

FIG. 4 is a graph of the effect of test Trichoderma strains on rice seedling root systems;

FIG. 5 is a colony graph after culturing at pH8.5 and 0% NaCl for 7d;

FIG. 6 is a colony map obtained by culturing at pH8.5 for 7d with 3% NaCl;

FIG. 7 is a colony graph obtained by culturing at 5% NaCl and pH8.5 for 7 days;

FIG. 8 is a graph of alfalfa seedling growth at the same time, wherein the left is 1% NaCl and the right is 1% NaCl + bacterial suspension;

FIG. 9 is a graph of alfalfa seedling growth at the same time, wherein the left is 2% NaCl, the right is 2% NaCl + bacterial suspension;

FIG. 10 is a graph of alfalfa seedling growth at the same time period, wherein the left is distilled water and the right is distilled water + bacterial suspension;

FIG. 11 is a graph showing the growth of rice seedlings at the same time, wherein the left is 1% of NaCl + bacterial suspension and the right is 1% of NaCl;

FIG. 12 is a graph of rice seedling growth at the same time period, wherein the left is 2% NaCl and the right is 2% NaCl + bacterial suspension;

FIG. 13 is a graph showing the growth of rice seedlings at the same time, wherein the left is distilled water and the right is distilled water + bacterial suspension;

FIG. 14 is a diagram of Trichoderma harzianum-17 strain morphology (40 times under microscope);

FIG. 15 is the morphology of Trichoderma harzianum M-17 colonies.

Detailed Description

The technical solutions of the present invention are further described in detail below with reference to specific examples, but the scope of the present invention is not limited to the following.

A saline-alkali tolerant Trichoderma strain, wherein the strain is Trichoderma harzianum-17, deposited in China center for type culture Collection with the following preservation number: CCTCC NO: m2018538.

Trichoderma harzianum-17 strain: the hypha is transparent, the wall is smooth, and the diameter is 1.7-6.4 μm; chlamydospores are spherical, unipartum, and intergrowth in the middle of hyphae. Conidia are very many on PDA culture medium, most are spherical, few are elliptical, most are 1.74-3.64 multiplied by 1.54-3.26 mu m, the wall is smooth, few are rough, and the conidia are semitransparent to light yellow green. Conidiophores are transparent, smooth in wall, straight or slightly curved, with enlarged base and thinner towards the top.

Specifically, the Trichoderma strain is tolerant to salt stress caused by NaCl concentration less than or equal to 5%; the trichoderma strains are tolerant to alkali stress caused by pH less than or equal to 8.5.

1. Screening and identification of saline-alkali-tolerant trichoderma strains

Collecting soil samples from the rhizosphere of sand-fixing plants in desert areas in Ningxia, separating and screening saline-alkali-tolerant trichoderma strains, and identifying the screened trichoderma strains through morphological observation, ITS sequence analysis and TEF sequence analysis to determine the classification status.

1.1 test materials

1.1.1 test soil samples

The method comprises the steps of randomly collecting rhizosphere soil of main sand fixing plants in Tenggeli desert and Wulanbu and desert areas in Ningxia, collecting soil samples with soil layers of 0-10cm close to plant roots, collecting about 40g of the soil samples, fully mixing the soil samples, removing stone grains and root systems, filling the soil samples into polyethylene bags, numbering the soil samples, bringing the soil samples back to a laboratory, immediately separating fungi, or temporarily storing the soil samples in a refrigerator with the temperature of 4 ℃ and then separating the soil samples.

1.1.2 test Medium

Martin's Bengal red medium, potato dextrose agar medium (PDA), potato Dextrose Broth (PDB)

1.1.3 test Agents

EDTA, tris-HCl, CTAB lysis buffer solution, 2-mercaptoethanol, extract liquor, TE buffer solution and primer

1.2 contents of the study

1.2.1 isolation of saline-alkali tolerant Trichoderma strains

A dilution plate method is adopted, namely 9-11g of soil sample is weighed and added into a conical flask containing 90ml of sterile water, and the conical flask is placed on a shaking table to oscillate for 10min, so that the soil sample is uniformly dispersed in a diluent to form a soil suspension. Diluting to 10-2 and 10-3 times by 10 times dilution method, respectively dripping 1ml of suspension into a sterilized culture dish with a diameter of 9cm, pouring 15ml of culture medium cooled to 40-45 deg.C, mixing with the suspension, solidifying, and culturing in 25 deg.C incubator for 5-7 days. 2 gradient dilutions were aspirated per soil sample, 3 replicates. The separation was performed using both a Martin Mengladesh red medium and a potato dextrose agar medium (PDA). When obvious trichoderma hyphae grow on the culture medium, adopting a sharp hyphae picking method to pick, transferring the trichoderma hyphae to a PDA slant culture medium, and carrying out purification culture at the temperature of 20-25 ℃.

1.2.2 screening of saline-alkali tolerant Trichoderma strains

The pH of the collected desert soil sample is between 8.4 and 8.7, so that the pH of the culture medium is fixed to 8.5, and the influence of NaCl on the growth of the strain is researched.

The selected Trichoderma strain was transferred to the center of a PDA plate containing different NaCl concentrations (0, 2%), each strain was repeated 3 times, cultured in a constant temperature incubator at 28 ℃ for 3-7d, and the colony diameter was measured. 5-10 trichoderma reesei with good salt tolerance effect are preliminarily screened out and then transferred to the center of a PDA (personal digital Assistant) plate containing different NaCl concentrations (0, 3 percent and 5 percent), each trichoderma reesei is repeated for 3 times, the trichoderma reesei is cultured in a constant-temperature incubator at 28 ℃ for 3-7 days, the sporulation condition of the trichoderma reesei is observed and recorded, and the colony diameter is measured.

1.2.3 screening of Trichoderma identification

And inoculating the screened trichoderma strains in a PDA (personal digital assistant) plate for culture, and carrying out colony morphology observation and microscopic morphology observation. Extracting trichoderma genome DNA by adopting a CTAB method, carrying out ITS and TEF fragment amplification reaction and sequence determination, submitting a sequence obtained after sequencing into local Blast of an ISTH website for sequence comparison, and identifying the species. The identification method of the saline-alkali tolerant trichoderma strains comprises the steps of extracting DNA of trichoderma harzianum M-17 by adopting a CTAB method, analyzing ITS sequences by adopting primers ITS1 and ITS4, analyzing TEF sequences by adopting primers EF1-728F and EF1-986R, submitting the sequences obtained after sequencing to local Blast of an ISTH website for sequence comparison, and identifying the species; the nucleotide sequence of the ITS1 is shown as SEQ ID No.1, the nucleotide sequence of the ITS4 is shown as SEQ ID No.2, the nucleotide sequence of the ITS is shown as SEQ ID No.3, the nucleotide sequence of the EF1-728F is shown as SEQ ID No.4, the nucleotide sequence of the EF1-986R is shown as SEQ ID No.5, and the nucleotide sequence of the TEF is shown as SEQ ID No. 6.

1.3 results of the study

1.3.1 isolation of saline-alkali tolerant Trichoderma strains

79 parts of soil samples are collected from the rhizosphere of sand-fixing plants in Tenggeli desert, wulanbu and desert areas in Ningxia, separated by a dilution plate method, and observed through colony morphology to obtain 43 trichoderma strains, wherein the soil sample information is shown in Table 1.

Tables 1 to 1: trichoderma soil sample information

Strain numbering

Original numbering

Collection ground

Vegetation

Strain numbering

Original numbering

Collection ground

Vegetation

m-1

W1-3

Umbelliferae and desert regions

m-23

TH1-P

Desert area in Ningxia

m-2

W1-10

Umbelliferae and grassland

m-25

TH1-M

Desert area in Ningxia

m-3

W1-35C

Umbelliferae and artificial interference area

m-26

TH1

Desert area in Ningxia

m-4

W1-51F

Umbelliferae and grassland

Caragana game

m-27

T22

Desert area in Ningxia

Phyllostachys Pubescens

m-5

W1-52G

Umbelliferae and grassland

Splendid achnatherum

m-28

T12

Desert area in Ningxia

Carnation bamboo

m-6

W1-35D

Umbelliferae and artificial interference area

m-29

TH2

Desert area in Ningxia

m-7

W1-28A

Umbelliferae and artificial interference area

m-30

T23

Desert area in Ningxia

Caragana microphylla

m-8

W1-55H

Umbelliferae and grassland

All-grass of Roughhaired Rabdosia

m-31

T11

Desert area in Ningxia

Ammopiptanthus mongolicus (lour.) Kuntze

m-9

W2-32-2

Umbelliferae and artificial interference area

m-32

T08

Desert area in Ningxia

Caragana microphylla

m-10

W2-33

Umbelliferae and artificial interference area

m-33

T09

Desert area in Ningxia

Caragana microphylla

m-11

W2-14

Umbelliferae and grassland

m-34

TS2

Desert area in Ningxia

m-12

W2-11

Umbelliferae and grassland

m-35

△T5

Desert area in Ningxia

White thorn

m-13

W2-2

Umbelliferae and desert regions

m-36

△TS1

Desert area in Ningxia

m-14

W2-1

Umbelliferae and desert regions

m-38

△T13

Within NingxiaDesert area

Artemisia desertorum

m-15

W2-5

Umbelliferae and desert region

m-39

W3-1A

Umbelliferae and desert region

m-16

W2-37

Umbelliferae and desert regions

Ammopiptanthus mongolicus

m-40

W3-5B1

Umbelliferae and desert regions

m-17

W2-32-1

Umbelliferae and artificial interference area

m-41

W3-5B2

Umbelliferae and desert region

m-18

W2-28

Umbelliferae and artificial interference area

m-42

W3-10C

Umbelliferae and grassland

m-19

T22-M

Desert area in Ningxia

Carnation bamboo

m-43

W3-44E1

Umbelliferae and grassland

Salt claw

m-20

T22-P

Desert area in Ningxia

Phyllostachys Pubescens

m-44

W3-51F2

Umbelliferae and artificial interference area

Splendid achnatherum

m-21

T11-2

Desert area in Ningxia

Camel hair

m-45

W3-52G

Umbelliferae and artificial interference area

Festuca arundinacea

m-22

T11-3

Desert area in Ningxia

Camel hair

1.3.2 preliminary screening of saline-alkali-tolerant Trichoderma strains

Tables 1 to 2:2% colony diameter of Trichoderma Strain at NaCl concentration

Tables 1-2 show the growth of the colonies of the Trichoderma strain at pH8.5, 2% NaCl concentration, i.e., the diameter of the colonies. As a result, it was found that the Trichoderma strain grew at pH8.5+0% by weight in the NaCl concentration, and that 74% of the Trichoderma strains grew normally to a diameter of 4cm or more after 7 days. At pH8.5+2% NaCl concentration, the Trichoderma strain grows slowly, after inoculation at 3d, m-4, m-6, m-8, m-10, m-16, m-30, no hypha grows from any of the 6 Trichoderma strains, and the difference between the colony diameter of the test strain and the cultured 3d CK is 0.25-3.25; after 5d of culture, all trichoderma strains grow out, the colony diameter is between 1.13 and 2.7cm, and the difference compared with CK cultured in 5d is between 0.43 and 4.44; after 7d of culture, the colony diameter of all trichoderma strains is between 1.55 and 3.88cm, and the difference compared with 5d of CK culture is between 0.68 and 4.87. Comprehensive analysis, the test Trichoderma strains finally screened 5 saline-alkali tolerant Trichoderma strains, namely m-2, m-17, m-26, m-33 and m-44, respectively, based on the standard that the difference between the concentration of NaCl at pH8.5+2 and the concentration of CK is small.

1.3.3 rescreening of saline-alkali tolerant Trichoderma strains

Tables 1 to 3: colony diameters of Trichoderma strains at different NaCl concentrations

Tables 1 to 4: sporulation of the selected strains at different NaCl concentrations

Note: "++++" spores cover the entire plate, "+++" indicates spores cover 3/4 of the colony, and "+" indicates spores cover 1/2 of the colony; "YG" yellow-green, "G" green, "LG" pale-green, "Y" yellow, "W" white.

Tables 1-3, tables 1-4 and FIGS. 5 to 7 show the growth of colonies of Trichoderma strains, i.e.the diameter of colonies and sporulation, at pH8.5 and different NaCl concentrations. From this, it was found that, at 5% NaCl concentration, none of the test strains grew after 3 days of culture, and m-17 strain grew best after 5 days of culture, the diameter of the colony was 0.83cm, and the diameter reached 1.45cm after 7 days of culture, which was significantly larger than that of the other strains, and the color of the hyphae was white; secondly, m-44 strains are adopted, the diameter of a colony after 5 days of culture is 0.67cm, the diameter after 7 days of culture is 1.13cm, and the color of hyphae is white; the m-26 strain grows slowly, the diameter of a bacterial colony is 0.37cm after 5 days of culture, the diameter of the bacterial colony is 0.45cm after 7 days of culture, and the color of hypha is white; while none of the other test strains m-2 and m-33 grew. Therefore, a trichoderma strain m-17 with good saline-alkali resistance is screened out.

1.3.4 identification of screening Trichoderma

Inoculating m-17 trichoderma strains to PDA to culture for 3d, and then carrying out colony morphology and microscopic observation, wherein the colony morphology is specifically described as follows:

bacterial colony: trichoderma harzianum strains were cultured on PDA plates in the dark at 25-28 ℃. After culturing for 1d, the front of the colony is in a white villous shape of aerial hyphae, and the back of the colony is colorless; after 2d of culture, the front of the colony generates light green conidia, the hyphae are loose and villiform, and the back of the colony is light green and ring-shaped; after 3d of culture, the bacterial colony grows over the whole flat plate, the hypha of the middle bacterial colony on the front surface is in a white villiform shape, the outer ring generates dark green conidium, and the back surface of the bacterial colony is green.

The strain is as follows: the hypha is transparent, the wall is smooth, and the diameter is 1.7-6.4 mu m; chlamydospores are spherical, unipartum, and intergrowth in the middle of hyphae. Conidia are very many on PDA culture medium, most are spherical, few are elliptical, most are 1.74-3.64 multiplied by 1.54-3.26 mu m, the wall is smooth, few are rough, and the conidia are semitransparent to light yellow green. Conidiophores are transparent, smooth in wall, straight or slightly curved, with enlarged base and thinner towards the top.

The strain morphology is in accordance with the Trichoderma harzianum (Trichoderma harzianum) description.

The sequencing result is submitted to Local Blast of the ISTH website for sequence alignment,

the method for identifying Trichoderma harzianum-17 comprises the steps of extracting DNA of Trichoderma harzianum M-17 by adopting a CTAB method, analyzing ITS sequences by adopting primers ITS1 and ITS4, analyzing TEF sequences by adopting primers EF1-728F and EF1-986R, submitting the sequences obtained after sequencing to local Blast of an ISTH website for sequence comparison, and comparing the sequences with the sequences of Trichoderma strain mode bacteria in an NCBI database by utilizing MEGA5.2.1 software to identify the Trichoderma harzianum (Trichoderma harzianum); the nucleotide sequence of the ITS1 is shown as SEQ ID No.1, the nucleotide sequence of the ITS4 is shown as SEQ ID No.2, the nucleotide sequence of the ITS is shown as SEQ ID No.3, the nucleotide sequence of the EF1-728F is shown as SEQ ID No.4, the nucleotide sequence of the EF1-986R is shown as SEQ ID No.5, and the nucleotide sequence of the TEF is shown as SEQ ID No. 6.

The morphological and molecular identification of the screened Trichoderma strain m-17 is Trichoderma harzianum (Trichoderma harzianum).

Tables 1 to 5: ITS sequence analysis of Trichoderma harzianum-17 strain

Tables 1 to 6: TEF sequence analysis of Trichoderma harzianum-17 Strain

2. Salt-decomposing and growth-promoting effect of saline-alkali-tolerant trichoderma strains on crops

The influence of the screened trichoderma strains on the germination and growth of the alfalfa and the rice is determined, the salt-decomposing and growth-promoting capacity of the trichoderma strains to be tested on the alfalfa and rice seedlings is determined, and a solid foundation is laid for the practical application of the saline-alkali tolerant trichoderma in the saline soil.

2.1 test materials

2.1.1 test strains: screened saline-alkali tolerant trichoderma (m-17)

2.1.2 test varieties: alfalfa (Imperial crown), rice (Ningjing 43)

2.1.3 preparation of spore suspension of Strain

Activating primary screening trichoderma, inoculating on PDA, culturing in 25 deg.C culture box, adding 1 drop Tween-80 and 5ml sterile water into the culture medium to obtain spore suspension, counting the concentration with blood counting plate to 1 × 10 ⁸ cfu/ml for use.

2.2 contents of the study

2.2.1 Effect of saline-alkali tolerant Trichoderma on crop seeds

Sterilizing the seeds by 5% NaClO solution for 5min, and washing with sterile water for 5 times. The seeds were then treated with the prepared spore suspension at a rate of 20. Mu.l/g. Placing the treated seeds in a culture dish spread with two layers of filter paper, and adding a solution containing 0% NaCl and 2% NaCl for moisture-keeping treatment. 30 per dish, 6 replicates. The culture medium was incubated in an incubator at 25 ℃ with the addition of an equal amount of sterile water as a control. And counting the number of the germinated seeds of the seeds day by day when the length of the radicle of the seeds exceeds half of the length of the seeds, stopping continuously counting until the germinated seeds do not increase, and calculating the germination rate, the germination index and the vitality index of the seeds.

Germination rate (%) = (total number of germinated seeds/total number of test seeds) × 100

Germination index = ∑ Gt/Dt

Wherein Gt is the number of germination in t time, and Dt is the corresponding number of germination days

Vigor index = germination index × average dry weight of individual plant

2.2.2 Effect of saline-alkali tolerant Trichoderma on crop growth

Selecting healthy seeds, sterilizing with 5% NaClO for 5min, and washing with sterile water for 5 times. And (3) filling sterilized sand into the plastic cups, respectively planting the sterilized seeds in the plastic cups, wherein 50 seeds per cup are subjected to test treatment when the seedlings grow to the period of two leaves and one heart. The test group was defined as the bacterial suspension, 1% NaCl + bacterial suspension, 2% NaCl + bacterial suspension, and the sterile water treatment was defined as the control. NaCl was applied to the soil as a salt stress treatment group, following the experimental setup, after which the bacterial suspension (1X 10) ⁸ cfu/ml) 50ml. Each treatment was repeated 6 times. The test was carried out in a greenhouse at 25 ℃ and 16h light. 30 seedlings were randomly picked from each treatment on day 30 after treatment. Cleaning, sucking dry water and measuring the growth index of the crop seedlings.

Plant height: measuring by a measuring tape; root-crown ratio: root dry weight/above ground dry weight; fresh weight: weighing the whole plant; dry weight: deactivating enzyme at 105 deg.C for 30min, drying at 70 deg.C to constant weight, and weighing; chlorophyll content: acetone ethanol mixed solution method.

Root morphology data: scanning a root system sample by using a root system scanner to obtain a digital image, and analyzing by using root system analysis system software to obtain root system morphological parameters including total root length, root surface area, root diameter, root volume and root tip number.

2.2.3 data processing

Excel is used for arranging data, DPS variance analysis is carried out, and a new repolarization difference method is adopted for carrying out multiple comparison.

2.3 results of the study and analysis thereof

2.3.1 Effect of saline-alkali tolerant Trichoderma on crop seeds

2.3.1.1 Effect of saline alkali tolerant Trichoderma on alfalfa seeds

Table 2-1: effect of test Trichoderma strains on alfalfa seed Activity under salt stress

Treatment of	Germination rate/%	Index of germination	Vitality index
				Distilled water	91.11	60.03	1.00
Distilled water + bacterial suspension	97.78	66.13	1.25
				2％NaCl	13.33	3.48	0.02
2% NaCl + bacterial suspension	38.89	15.91	1.21

As can be seen from Table 2-1 and FIG. 1, the different treatments had a more pronounced effect on the germination of alfalfa seeds. After the distilled water and the bacterial suspension are treated, the germination rate, the germination index and the vitality index can be obviously improved by 7.32 percent, 10.16 percent and 25 percent respectively compared with the distilled water treatment; 2 percent, all related indexes of alfalfa seed germination are obviously lower than those of distilled water treatment under the stress of NaCl, and all indexes have a rising trend through seed soaking of test trichoderma suspension. 2% germination, germination index and viability index of the NaCl + bacterial suspension treatment, all improved 191.75%, 357.18% and 5950% over 2% NaCl treatment. The germination rate reflects the germination capacity of the seeds; the germination index can reflect the germination speed to a certain extent, and the seed vigor index can comprehensively reflect the seed quality. The result shows that the test trichoderma suspension seed soaking can effectively relieve the inhibition effect of salt stress on alfalfa seed germination, and has the effect of promoting seed germination.

2.3.1.2 Effect of saline-alkali tolerant Trichoderma on Rice seeds

Tables 2 to 2: effect of test Trichoderma strains on Rice seed vigor under salt stress

Treatment of	Germination rate	Index of germination	Vitality index
				Distilled water	91.11	43.24	2.76
Distilled water + bacterial suspension	95.56	45.47	3.29
				2％NaCl	24.44	2.1	0.14
2% NaCl + bacterial suspension	64.44	8.62	0.67

As can be seen from tables 2-2 and FIG. 2, the different treatments had a more pronounced effect on the germination of rice seeds. After the distilled water and the bacterial suspension are treated, the germination rate, the germination index and the vitality index can be obviously improved, and are respectively improved by 4.88 percent, 5.16 percent and 19.20 percent compared with the distilled water treatment; 2% under NaCl stress, the indexes related to rice seed germination are all obviously lower than those of distilled water treatment, and all the indexes have a rising trend through seed soaking of test trichoderma suspension. 2% germination percentage, germination index and viability index of the NaCl + bacterial suspension treatment, all increased 163.67%, 310.48% and 378.57% compared to 2% NaCl treatment. The results show that the test trichoderma suspension seed soaking can improve rice seed germination and relieve the inhibition effect of salt stress.

2.3.2 Effect of saline-alkali tolerant Trichoderma on crop growth

2.3.2.1 Effect of saline-alkali tolerant Trichoderma on alfalfa growth

2.3.2.1.1 Effect of saline-alkali tolerant Trichoderma on alfalfa seedling growth

Tables 2 to 3: effect of test Trichoderma strains on alfalfa seedling growth

As can be seen from tables 2-3 and fig. 8 to 10: different treatments had a more pronounced effect on alfalfa seedling growth. After inoculation and culture for 30 days, seedlings are taken out for measurement, and results show that after treatment of distilled water and bacterial suspension, compared with the treatment of distilled water, the plant height, fresh weight, dry weight, root-crown ratio and chlorophyll content are respectively improved by 14.66%, 5.46%, 8.18%, 9.52% and 7.5%, and the difference is obvious; under the stress of NaCl, all relevant indexes of the growth of the alfalfa seedlings are obviously lower than those of distilled water treatment, and the growth of the alfalfa seedlings is hindered along with the increase of NaCl concentration. After the test trichoderma suspension is added, all indexes have a rising trend. 1% NaCl + suspension treated plant height, fresh weight, dry weight, root-cap ratio and chlorophyll content were all improved by 43.02%, 57.21%, 22.89%, 31.03% and 38.10% over 1% NaCl treatment, and 2% NaCl + suspension treated plant height, fresh weight, dry weight, root-cap ratio and chlorophyll content were all improved by 36.70%, 22.11%, 9.46%, 12% and 120.53% over 2% NaCl treatment. The results show that the tested trichoderma suspension can promote the growth of alfalfa seedlings and relieve the salt stress inhibition effect.

2.3.2.1.2 Effect of saline-alkali tolerant Trichoderma on the agronomic traits of alfalfa seedling underground

Tables 2 to 4: influence of test Trichoderma strains on root system of alfalfa seedling

Treatment of	Total root length (cm)	Surface area (cm) 2 )	Average diameter (mm)	Root volume (cm) 3 )	Root tip number (number)
						Distilled water	54.53±7.38b	10.14±0.46b	0.301±0.0134a	0.030±0.005b	52±1.7bc
Distilled water + bacterial suspension	86.02±2.79a	11.83±0.68a	0.280±0.011a	0.039±0.002ab	58±1.5b
						1％NaCl	34.97±8.25c	6.91±1.60c	0.306±0.013a	0.016±0.006c	31±0.8cd
1% NaCl + suspension	77.09±5.51a	10.21±0.07b	0.297±0.011a	0.041±0.012ab	74±0.9ab
						2％NaCl	25.48±5.36c	6.19±1.22c	0.297±0.055a	0.008±0.003c	27±1.1d
2% NaCl + bacterial suspension	61.85±12.19b	9.04±0.22b	0.293±0.008a	0.044±0.010a	81±1.0a

As can be seen from tables 2-4 and FIG. 3, after 30 days of inoculation and culture, sampling and scanning are performed to obtain images of the root system of each treated alfalfa, and morphological parameters of the root system are obtained by analyzing with root system analysis software, and the results show that different treatments have obvious influence on the development of the root system of the alfalfa seedling. After the distilled water and the bacterial suspension are treated, the root system is developed, the total root length and the root surface area are respectively improved by 57.75 percent and 16.67 percent compared with the distilled water treatment, the difference is obvious, the root volume and the number of root tips are respectively improved by 7.5 percent, 30 percent and 11.54 percent, but the difference is not obvious; under the stress of NaCl, all relevant indexes of the root system of the alfalfa seedlings are obviously lower than those of distilled water treatment, and the growth of the rhizosphere of the alfalfa seedlings is hindered along with the increase of the concentration of the NaCl. After the test trichoderma suspension is added, all indexes have a rising trend. The 1% NaCl + bacterial suspension treatment total root length, root surface, root volume and root tip number were all increased by 120.45%, 47.76%, 156.25% and 138.71% over the 1% NaCl treatment, and the 2% NaCl + bacterial suspension treatment total root length, root surface, root volume and root tip number were all increased by 142.74%, 46.04%, 450% and 200% over the 2% NaCl treatment. The result shows that the tested trichoderma suspension can effectively relieve the inhibition effect of salt stress on the roots of alfalfa seedlings and promote the growth of the roots.

2.3.2.2 Effect of saline-alkali tolerant Trichoderma on Rice growth

2.3.2.2.1 Effect of saline-alkali tolerant Trichoderma on growth of Rice seedlings

Tables 2 to 5: effect of test Trichoderma strains on growth of Rice seedlings

As can be seen from tables 2-5 and FIGS. 11 to 13, the different treatments had a more pronounced effect on the growth of rice seedlings. After inoculation and culture for 30d, seedlings are taken out for measurement, and the results show that after treatment of distilled water and bacterial suspension, compared with the treatment of distilled water, the plant height, the fresh weight, the dry weight and the chlorophyll content are respectively improved by 52.66%, 28.24%, 24.93% and 10.27%, the difference is obvious, but the root-crown ratio is not different; under the stress of NaCl, all relevant indexes of the growth of the alfalfa seedlings are obviously lower than those of distilled water treatment, and the growth of the alfalfa seedlings is hindered along with the increase of NaCl concentration. After the test trichoderma suspension is added, all indexes have a rising trend. 1% NaCl + strain height, fresh weight, dry weight and chlorophyll content of the strain suspension treatment were all increased by 18.75%, 21.68%, 24.99% and 66.57% from 1% NaCl treatment, 2% NaCl + strain height, fresh weight, dry weight and chlorophyll content of the strain suspension treatment were all increased by 10.40%, 2.80%, 12.04% and 63.48% from 2% NaCl treatment, the root-cap ratio was reduced and was not different. The results show that the tested trichoderma suspension can promote the growth of rice seedlings, improve the chlorophyll content, increase photosynthesis and relieve the salt stress inhibition.

2.3.2.2.2 Effect of saline-alkali tolerant Trichoderma on the agronomic traits of underground parts of Rice seedlings

Tables 2 to 6: effect of test Trichoderma strains on Rice seedling root System

Treatment of	Total root length (cm)	Surface area (cm) 2 )	Average diameter (mm)	Root volume (cm) 3 )	Root tip number (number)
						Distilled water	78.09±16.41a	10.72±1.64a	0.227±0.021b	0.043±0.003a	953±3.39ab
Distilled water + bacterial suspension	91.57±8.91a	11.22±1.18a	0.232±0.012b	0.061±0.014a	1272±1.50a
						1％NaCl	56.73±12.44b	9.85±1.40a	0.220±0.012b	0.042±0.018a	553±3.20b
1% NaCl + suspension	83.16±6.26a	11.64±0.80a	0.288±0.034a	0.047±0.003a	1079±4.66a
						2％NaCl	40.39±17.08b	11.67±1.27a	0.207±0.020b	0.039±0.008a	461±1.51b
2% NaCl + bacterial suspension	73.73±2.41a	12.00±0.16a	0.227±0.018b	0.046±0.020a	829±3.18ab

As can be seen from tables 2-6 and FIG. 4, after inoculation and cultivation for 30d, sampling and scanning are carried out to obtain images of root systems of rice seedlings to be treated, and morphological parameters of the root systems are obtained by analyzing through root system analysis software. H ₂ After the O + bacterial suspension is treated, H is mixed with the O + bacterial suspension ₂ Compared with the O treatment, the total root length, the root surface, the average diameter, the root volume and the number of root tips are respectively improved by 17.26 percent, 4.66 percent, 2.20 percent, 41.86 percent and 33.47 percent, but the difference is not obvious; under the stress of NaCl, all related indexes of the root system of the rice seedling are obviously lower than H ₂ And (4) O treatment, wherein the rhizosphere growth of the rice seedlings is hindered along with the increase of the NaCl concentration. After the test trichoderma suspension is added, all indexes have a rising trend. 1% NaCl + suspension treatment the total root length, mean diameter and root tip number were improved by 46.59%, 30.90% and 77.03% over 1% NaCl treatment with significant differences, and the root surface and root volume were also improved by 18.17% and 11.9% over 1% NaCl treatment with insignificant differences. 2% NaCl + suspension treatment the total root length was increased 82.55% over 2% NaCl treatment with significant differences, the root surface, mean diameter, root volume and number of root tips were increased 2.83%, 7.78%, 11.86% and 79.83% over 2% NaCl treatment, but the differences were not significant. The result shows that the tested trichoderma suspension can relieve the inhibition effect of salt stress on the roots of rice seedlings and has a certain promotion effect on the growth of the roots.

3. Summary of the invention

79 parts of soil samples are collected from the rhizosphere of sand-fixing plants in Tenggeli desert, wulanbu and desert areas in Ningxia, separated by a dilution plate method, and observed through colony morphology to obtain 43 trichoderma strains. The growth diameter of the colony of the Trichoderma strain is investigated under the conditions of pH8.5 and NaCl concentration of 2 percent, and 5 saline-alkali resistant Trichoderma strains are preliminarily screened out, wherein the concentration is m-2, m-17, m-26, m-33 and m-44 respectively. The growth diameter and spore production condition of trichoderma strains are examined under the conditions of pH8.5 and different NaCl (0, 3 percent and 5 percent) concentrations, and finally a trichoderma strain m-17 with good saline-alkali resistance is screened out. Through morphological and molecular identification, the saline-alkali tolerant Trichoderma harzianum strain is known to be Trichoderma harzianum.

Through determining indexes of the saline-alkali tolerant trichoderma strains on germination of alfalfa and rice seeds, seedling growth, root development and the like, the result shows that the test trichoderma suspension seed soaking can effectively relieve the inhibition effect of salt stress on germination of alfalfa and rice seeds, and has the effect of promoting seed germination. Meanwhile, the growth of alfalfa and rice seedlings can be promoted, and the salt stress inhibition effect can be relieved. Salt stress affects important life processes such as plant photosynthesis, protein synthesis and energy metabolism. The test shows that the germination and seedling growth of alfalfa and rice seeds are severely inhibited under the condition of salt stress. By soaking seeds in the tested trichoderma suspension, the harm of salt stress to the germination of alfalfa and rice seeds can be effectively reduced, the photosynthesis of the plant chlorophyll is protected by promoting the synthesis of the plant chlorophyll, the normal growth and seedling growth of the plant are maintained, and the chlorophyll content is increased.

The foregoing is illustrative of the preferred embodiments of the present invention, and it is to be understood that the invention is not limited to the precise form disclosed herein and is not to be construed as limited to the exclusion of other embodiments, and that various other combinations, modifications, and environments may be used and modifications may be made within the scope of the concepts described herein, either by the above teachings or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> Ningxia academy of agriculture and forestry, institute of plant protection

<120> saline-alkali tolerant trichoderma strain, separation and screening method, culture method, application and use method

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<213> 10

<400> 10

ttctgtaggg gtgaacctgc ggagggatca ttaccgagtt tacaactccc aaacccaatg 60

tgaacgttac caaactgttg cctcggcggg atctctgccc cgggtgcgtc gcagccccgg 120

accaaggcgc ccgccggagg accaaccaaa actctttttg tataccccct cgcgggtttt 180

ttataatctg agccttctcg gcgcctctcg taggcgtttc gaaaatgaat caaaactttc 240

aacaacggat ctcttggttc tggcatcgat gaagaacgca gcgaaatgcg ataagtaatg 300

tgaattgcag aattcagtga atcatcgaat ctttgaacgc acattgcgcc cgccagtatt 360

ctggcgggca tgcctgtccg agcgtcattt caaccctcga acccctccgg ggggtcggcg 420

ttggggatcg gccctgcctc ttggcggtgg ccgtctccga aatacagtgg cggtctcgcc 480

gcagcctctc ctgcgcagta gtttgcacac tcgcatcggg agcgcggcgc gtccacagcc 540

gttaaacacc caacttctga aatgttgacc tcggatcagg taggaatacc cgctgaactt 600

aagcatatca ataagccgga ggaaa 625

<210> 11

<211> 646

<212> DNA

<213> 11

<400> 11

ggaagtaaaa agtcgtaaca aggtctccgt tggtgaacca gcggagggat cattaccgag 60

tttacaactc ccaaacccaa tgtgaacgtt accaaactgt tgcctcggcg ggatctctgc 120

cccgggtgcg tcgcagcccc ggaccaaggc gcccgccgga ggaccaacca aaactctttt 180

tgtatacccc ctcgcgggtt ttttataatc tgagccttct cggcgcctct cgtaggcgtt 240

tcgaaaatga atcaaaactt tcaacaacgg atctcttggt tctggcatcg atgaagaacg 300

cagcgaaatg cgataagtaa tgtgaattgc agaattcagt gaatcatcga atctttgaac 360

gcacattgcg cccgccagta ttctggcggg catgcctgtc cgagcgtcat ttcaaccctc 420

gaacccctcc ggggggtcgg cgttggggat cggccctgcc tcttggcggt ggccgtctcc 480

gaaatacagt ggcggtctcg ccgcagcctc tcctgcgcag tagtttgcac actcgcatcg 540

ggagcgcggc gcgtccacag ccgttaaaca cccaacttct gaaatgttga cctcggatca 600

ggtaggaata cccgctgaac ttaagcatat caataagcgg gaggaa 646

<210> 12

<211> 643

<212> DNA

<213> 12

<400> 12

ggaagtaaaa gtcgtaacaa gctctccgtt ggtgaaccag cggagggatc attaccgagt 60

ttacaactcc caaacccaat gtgaacgtta ccaaactgtt gcctcggcgg gatctctgcc 120

ccgggtgcgt cgcagccccg gaccaaggcg cccgccggag gaccaaccaa aactcttttt 180

gtataccccc tcgcgggttt tttataatct gagccttctc ggcgcctctc gtaggcgttt 240

cgaaaatgaa tcaaaacttt caacaacgga tctcttggtt ctggcatcga tgaagaacgc 300

agcgaaatgc gataagtaat gtgaattgca gaattcagtg aatcatcgaa tctttgaacg 360

cacattgcgc ccgccagtat tctggcgggc atgcctgtcc gagcgtcatt tcaaccctcg 420

aacccctccg gggggtcggc gttggggatc ggccctgcct cttggcggtg gccgtctccg 480

aaatacagtg gcggtctcgc cgcagcctct cctgcgcagt agtttgcaca ctcgcatcgg 540

gagcgcggcg cgtccacagc cgttaaacac ccaacttctg aaatgttgac ctcggatcag 600

gtaggaatac ccgctgaact taagcatatc aataagcgga gga 643

<210> 13

<211> 420

<212> DNA

<213> 13

<400> 13

acctgcggag ggatcattac cgagtttaca actcccaaac ccaatgtgaa cgttaccaaa 60

ctgttgcctc ggcgggatct ctgccccggg tgcgtcgcag ccccggacca aggcgcccgc 120

cggaggacca accaaaactc tttttgtata ccccctcgcg ggttttttat aatctgagcc 180

ttctcggcgc ctctcgtagg cgtttcgaaa atgaatcaaa actttcaaca acggatctct 240

tggttctggc atcgatgaag aacgcagcga aatgcgataa gtaatgtgaa ttgcagaatt 300

cagtgaatca tcgaatcttt gaacgcacat tgcgcccgcc agtattctgg cgggcatgcc 360

tgtccgagcg tcatttcaac cctcgaaccc ctccgggggg tcggcgttgg ggatcggccc 420

<210> 14

<211> 611

<212> DNA

<213> 14

<400> 14

ccgtaggtga acctgcggag ggatcattac cgagtttaca actcccaaac ccaatgtgaa 60

cgttaccaaa ctgttgcctc ggcgggatct ctgccccggg tgcgtcgcag ccccggacca 120

aggcgcccgc cggaggacca accaaaactc tttttgtata ccccctcgcg ggttttttta 180

taatctgagc cttctcggcg cctctcgtag gcgtttcgaa aatgaatcaa aactttcaac 240

aacggatctc ttggttctgg catcgatgaa gaacgcagcg aaatgcgata agtaatgtga 300

attgcagaat tcagtgaatc atcgaatctt tgaacgcaca ttgcgcccgc cagtattctg 360

gcgggcatgc ctgtccgagc gtcatttcaa ccctcgaacc cctccggggg gtcggcgttg 420

gggatcggcc ctgcctcttg gcggtggccg tctccgaaat acagtggcgg tctcgccgca 480

gcctctcctg cgcagtagtt tgcacactcg catcgggagc gcggcgcgtc cacagccgtt 540

aaacacccaa cttctgaaat gttgacctcg gatcaggtag gaatacccgc tgaacttaag 600

catatcaata a 611

<210> 15

<211> 340

<212> DNA

<213> 15

<400> 15

catcgagaag ttcgagaagg taagcttcaa ctgattttcg cctcgattcc tcttctttca 60

tattcaattg tgcccgacaa ttcagagaat tttcgtgtcg acaattttca tcaccccgct 120

ttccattacc cctcctttgc agcgacgcaa attttttttg ctgccgtttg agttttagtg 180

gggttctctg tgcaacccca ctagctcact gctttttttg tgcttcacac tcacttccaa 240

gtcatcattc aacgtgctct gtgtctttgg tcattcaacg atgctaacca cttttccatc 300

aataggaagc cgccgaactc ggtaagggtt ccttcaagta 340

<210> 16

<211> 568

<212> DNA

<213> 16

<400> 16

cggtggtatc gaccgtcgta ccatcgagaa gttcgagaag gtaagcgtca actgattttc 60

gcctcgattc ctcttctttc atattcaatt gtgcccgaca attcttcaga gactttttgg 120

gtcgacaatt tttcgtcacc ccgctttcca ttacccctcc tttgcagcga cgcaaatttt 180

ttttgctgcc gtttgatttt tagtggggtt ctctgtgcaa ccccactagc tcactgcttt 240

ttttgtgctt cattcacttc ccagtcatca ttcaacgtgc tctgtgtctt tggttattca 300

acgatgctaa ccacttttcc atcaatagga agccgccgaa ctcggcaagg gttccttcaa 360

gtacgcttgg gttcttgaca agctcaaggc cgagcgtgag cgtggtatca ccatcgacat 420

tgctctgtgg aagttcgaga ctcccaagta ctatgtcacc gtcattggta tgtctacttc 480

atcaacttca tgctgcaatt gcaacccagt gctaacaggc aattcacaga cgctcccggc 540

caccgtgatt tcatcaagaa catgatca 568

<210> 17

<211> 644

<212> DNA

<213> 17

<400> 17

tcttctacca gtgcggtggt atcgaccgtc gtaccatcga gaagttcgag aaggtaagct 60

tcaactgatt ttcgcctcga ttcctctttc atattcaatt gtgcccgaca atccttcaga 120

gaattttcgt gtcgacaatt tttcatcacc ccgctttcca ttacccctcc tttgcagcga 180

cgcaaatttt ttttgctgtc gtttgatttt tagtggggtt ctctgtgcaa ccccactagc 240

tcactgcttt tcttgtgctt cactcacttc ccagtcatca ttcaacgtgc tgtgtgtctt 300

tggtcattca acgatgctaa ccacttttcc atcaatagga agccgccgaa ctcggcaagg 360

gttccttcaa gtacgcttgg gttcttgaca agctcaaggc cgagcgtgag cgtggtatca 420

ccatcgacat tgctctgtgg aagttcgaga ctcccaagta ctatgtcacc gtcattggta 480

tgtctacttc atcaacttca tgctgcaatt gcaacccagt gctaacaggc aattcacaga 540

cgctcccggc caccgtgatt tcatcaagaa catgatcact ggtacttccc aggccgattg 600

cgctatcctc atcattgccg ccggtactgg tgagttcaag gctg 644

<210> 18

<211> 528

<212> DNA

<213> 18

<400> 18

atcgagaagt tcgagaaggt aagcttcaac tgattttcgc ctcgattctt cctccttcac 60

attcaattgt gctcgacaat tctgcagaga attttcgtgt cgacaatttt tcatcacccc 120

gctttccatt acccctcctt tgcagcgacg caaatttttt ttgctgccgt ttgattttta 180

gtggggttct ttgtgcaacc ccactagctc actgcttttt tttgtgcttc actctcactt 240

cccagccatc attcaacgtg ctctgtgtct ttggtcattc agcgatgcta accacttttc 300

catcaatagg aagccgccga actcggcaag ggttccttca agtacgcttg ggttcttgac 360

aagctcaagg ccgagcgtga gcgtggtatc accatcgaca ttgctctgtg gaagttcgag 420

actcccaagt actatgtcac cgtcattggt atgtcttcat caatctcatg gtgcaactgc 480

gagctagtgc taacatgcaa ttcacagacg ctcccggcca ccgtgatt 528

<210> 19

<211> 589

<212> DNA

<213> 19

<400> 19

taagcgtcaa ctgattttcg cctcgattcc tcttctttca tattcaattg tgcccgacaa 60

ttcttcagag actttttggg tcaacaattt ttcgtcaccc cgctttccat tacccctcct 120

ttgcagcgac gcaaattttt tttgctgccg tttgattttt agtggggttc tctgtgcaac 180

cccactagct cactgctttt tttgtgcttc attcacttcc cagtcatcat tcaacgtgct 240

ctgtgtcttt ggttattcaa cgatgctaac cacttttcca tcaataggaa gccgccgaac 300

tcggcaaggg ttccttcaag tacgcttggg ttcttgacaa gctcaaggcc gagcgtgagc 360

gtggtatcac catcgacatt gctctgtgga agttcgagac tcccaagtac tatgtcaccg 420

tcattggtat gtctacttca tcaacttcat gctgcaattg caacccagtg ctaacaggca 480

attcacagac gctcccggcc accgtgattt catcaagaac atgatcactg gtacttccca 540

ggccgattgc gctatcctca tcattgccgc cggtactggt gagttcgag 589

<210> 20

<211> 344

<212> DNA

<213> 20

<400> 20

catcgagaag ttcgagaagg taagcttcaa ctgattttcg cctcgattct tcctccttca 60

cattcaattg tgcccgacaa ttctgcagag aattttcgtg tcgacaattt ttcatcaccc 120

cgctttccgt tacccctcct ttgcagcgac gcaaaatttt ttttgctgtc gtctggtttt 180

tagtggggtt ctctgtgcaa ccccactagc tcactgcttt ttcctgcttc actctcactt 240

cctcgtcatc attcaacacg ctctgtgtct ttggtcattc agcgatgcta accacttttc 300

catcaatagg aagccgccga actcggtaag ggttccttca agta 344

<210> 21

<211> 771

<212> DNA

<213> 21

<400> 21

tttcgtacgt atcatcctgt tccctcacgt cggcatcatt cgccgctctg attcccaaac 60

gcttgtgcta accatcaact tctaggggtg cgtattccat caattatctt gaatgagatc 120

gaccgaacac aatactgact tgctacaaca gccacgtcga ctccggaaag tcgaccaccg 180

taagttgcac cctctcttgc tccgatatca aacgtcgttt gatggggaca catactcttg 240

aacacagggc taatcattca tcatacagac cggtcacttg atctaccagt gcggtggtat 300

cgaccgtcgt accatcgaga agttcgagaa ggtaagcttc aactgatttt cgcctcgatt 360

cttcctcctc cacattcaat tgtgcccgac aattctgcag agaattttcg tgtcgacaat 420

ttttcatcac cccgctttcc attacccctc ctttgcagcg acgcaaattt ttttttgctg 480

ccgtttgatt tttagtgggg ttctttgtgc aaccccacta gctcactgct tttttttgtg 540

cttcactctc acttcccagc catcattcaa cgtgctctgt gtctttggtc attcagcgat 600

gctaaccact ttttccatca ataggaagcc gccgaactcg gcaagggttc cttcaagtac 660

gcttgggttc ttgacaagct caaggccgag cgtgagcgtg gtatcaccat cgatattgct 720

ctgtggaagt tcgagactcc caggtactat gtcaccgtca ttggtatgcc c 771

<210> 22

<211> 906

<212> DNA

<213> 22

<400> 22

aagactcaca tcaacgtggt cgttatcgta cgtatcatcc tttctcacgt cggcatcatt 60

cgccggtctg attctcaaac ttatgctaac catcgccttc taggggtgcg tattccatca 120

atcatcttga atgagatcga tcgaacacaa tactgacttg ctacaacagc cacgtcgact 180

ccggaaagtc gaccaccgta agttgcaccc tttcttgctc cgatatcaaa cgtcgtttga 240

tgcgggacat ctactcttga acacagggct aaccattcat catacagacc ggtcacttga 300

tctaccagtg cggtggtatc gaccgtcgta ccatcgagaa gttcgagaag gtaagcttca 360

actgattttc gcctcgattc ctcttctttc atattcaatt gtgcccgaca attcagagaa 420

ttttcgtgtc gacaattttc atcaccccgc tttccattac ccctcctttg cagcgacgca 480

aatttttttt gctgccgttt gagttttagt ggggttctct gtgcaacccc actagctcac 540

tgcttttttt gtgcttcaca ctcacttcca agtcatcatt caacgtgctc tgtgtctttg 600

gtcattcaac gatgctaacc acttttccat caataggaag ccgccgaact cggcaagggt 660

tccttcaagt acgcttgggt tcttgacaag ctcaaggccg agcgtgagcg tggtatcacc 720

atcgacattg ctctgtggaa gttcgagact cccaagtact atgtcaccgt cattggtatg 780

tctacttcat caacttcatg ctgcaattgc aacccagtgc taacaggcaa ttcacagacg 840

ctcccggcca ccgtgatttc atcaagaaca tgatcactgg tacttcccag gccgattgcg 900

ctatcc 906

<210> 23

<211> 906

<212> DNA

<213> 23

<400> 23

aagactcaca tcaacgtggt cgttatcgta cgtatcatcc tttctcacgt cggcatcatt 60

cgccggtctg attctcaaac ttatgctaac catcgccttc taggggtgcg tattccatca 120

atcatcttga atgagatcga tcgaacacaa tactgacttg ctacaacagc cacgtcgact 180

ccggaaagtc gaccaccgta agttgcaccc tttcttgctc cgatatcaaa cgtcgtttga 240

tgcgggacat ctactcttga acacagggct aaccattcat catacagacc ggtcacttga 300

tctaccagtg cggtggtatc gaccgtcgta ccatcgagaa gttcgagaag gtaagcttca 360

actgattttc gcctcgattc ctcttctttc atattcaatt gtgcccgaca attcagagaa 420

ttttcgtgtc gacaattttc atcaccccgc tttccattac ccctcctttg cagcgacgca 480

aatttttttt gctgccgttt gagttttagt ggggttctct gtgcaacccc actagctcac 540

tgcttttttt gtgcttcaca ctcacttcca agtcatcatt caacgtgctc tgtgtctttg 600

gtcattcaac gatgctaacc acttttccat caataggaag ccgccgaact cggcaagggt 660

tccttcaagt acgcttgggt tcttgacaag ctcaaggccg agcgtgagcg tggtatcacc 720

atcgacattg ctctgtggaa gttcgagact cccaagtact atgtcaccgt cattggtatg 780

tctacttcat caacttcatg ctgcaattgc aacccagtgc taacaggcaa ttcacagacg 840

ctcccggcca ccgtgatttc atcaagaaca tgatcactgg tacttcccag gccgattgcg 900

ctatcc 906

<210> 24

<211> 783

<212> DNA

<213> 24

<400> 24

gcatcattcg ccggtctgat tctcaaactt atgctaacca tcgccttcta ggggtgcgta 60

ttccatcaat catcttgaat gagatcgatc gaacacaata ctgacttgct acaacagcca 120

cgtcgactcc ggaaagtcga ccaccgtaag ttacaccctt tcttgctccg atatcaaacg 180

tcgtttgatg cgggacatct actcttgaac acagggctaa ccattcatca tacagaccgg 240

tcacttgatc taccagtgcg gtggtatcga ccgtcgtacc atcgagaagt tcgagaaggt 300

aagcttcaac tgattttcgc ctcgattcct cttctttcat attcaattgt gcccgacaat 360

tcagagaatt ttcgtgtcga caattttcat caccccgctt tccattaccc ctcctttgca 420

gcgacgcaaa ttttttttgc tgccgtttga gttttagtgg ggttctctgt gcaaccccac 480

tagctcactg ctttttttgt gcttcacact cacttccaag tcatcattca acgtgctctg 540

tgtctttggt cattcaacga tgctaaccac ttttccatca ataggaagcc gccgaactcg 600

gcaagggttc cttcaagtac gcttgggttc ttgacaagct caaggccgag cgtgagcgtg 660

gtatcaccat cgacattgct ctgtggaagt tcgagactcc caagtactat gtcaccgtca 720

ttggtatgtc tacttcatca acttcatgct gcaattgcaa cccagtgcta acaggcaatt 780

cac 783

<210> 25

<211> 877

<212> DNA

<213> 25

<400> 25

tcacatcaac gtggtcgtta tcgtacgtat catcctttct cacgtcggca tcattcgccg 60

gtctgattct caaacttgtg ctaatcatcg ccttctaggg gtgcgtattc catcaatcat 120

cttgaatgag atcgatcgaa cacaatactg acttgctaca acagccacgt cgactccgga 180

aagtcgacca ccgtaagtta caccctctct tgctccgata tcaaacgtcg tttgatgcgg 240

gacatctact cttgaacaca gggctaacca ttcatcatac agaccggtca cttgatctac 300

cagtgcggtg gtatcgaccg tcgtaccatc gagaagttcg agaaggtaag cttcaactga 360

ttttcgcctc gattcctctt ctttcatatt caattgtgcc cgacaattct tcagactttt 420

tgggtcgaca atttttcgtc accccgcttt ccattacccc tcctttgcag cgacgcaaat 480

tttttttgct gccgtttgat ttttagtggg gttctctgtg caaccccact agctcactgc 540

tttttttgtg cttcattcac ttcccagtca tcattcaacg tgctctgtgt ctttggttat 600

tcaacgatgc taaccacttt tccatcaata ggaagccgcc gaactcggca agggttcctt 660

caagtacgct tgggttcttg acaagctcaa ggccgagcgt gagcgtggta tcaccatcga 720

cattgctctg tggaagttcg agactcccaa gtactatgtc accgtcattg gtatgtctac 780

ttcatcaact tcatgctgca attgcaaccc agtgctaaca ggcaattcac agacgctccc 840

ggccaccgtg atttcatcaa gaacatgatc actggta 877

<210> 26

<211> 338

<212> DNA

<213> 26

<400> 26

atcgagaagt tcgagaaggt aagcttcaac tgattttcgc ctcgattcct cttctttcat 60

attcaattgt gcccgacaat tcagagaatt ttcgtgtcga caattttcat caccccgctt 120

tccattaccc ctcctttgca gcgacgcaaa ttttttttgc tgccgtttga tttttagtgg 180

ggttctctgt gcaacccact agctcactgc tttttttgtg cttcacactc acttccaagt 240

catcattcaa cgtgctctgt gtctttggtc attcaacgat gctaaccact tttccatcaa 300

taggaagccg ccgaactcgg taagggttcc ttcaagta 338

<210> 27

<211> 906

<212> DNA

<213> 27

<400> 27

aagactcaca tcaacgtggt cgttatcgta cgtatcatcc tttctcacgt cggcatcatt 60

cgccggtctg attctcaaac ttatgctaac catcgccttc taggggtgcg tattccatca 120

atcatcttga atgagatcga tcgaacacaa tactgacttg ctacaacagc cacgtcgact 180

ccggaaagtc gaccaccgta agttgcaccc tttcttgctc cgatatcaaa cgtcgtttga 240

tgcgggacat ctactcttga acacagggct aaccattcat catacagacc ggtcacttga 300

tctaccagtg cggtggtatc gaccgtcgta ccatcgagaa gttcgagaag gtaagcttca 360

actgattttc gcctcgattc ctcttctttc atattcaatt gtgcccgaca attcagagaa 420

ttttcgtgtc gacaattttc atcaccccgc tttccattac ccctcctttg cagcgacgca 480

aatttttttt gctgccgttt gagttttagt ggggttctct gtgcaacccc actagctcac 540

tgcttttttt gtgcttcaca ctcacttcca agtcatcatt caacgtgctc tgtgtctttg 600

gtcattcaac gatgctaacc acttttccat caataggaag ccgccgaact cggcaagggt 660

tccttcaagt acgcttgggt tcttgacaag ctcaaggccg agcgtgagcg tggtatcacc 720

atcgacattg ctctgtggaa gttcgagact cccaagtact atgtcaccgt cattggtatg 780

tctacttcat caacttcatg ctgcaattgc aacccagtgc taacaggcaa ttcacagacg 840

ctcccggcca ccgtgatttc atcaagaaca tgatcactgg tacttcccag gccgattgcg 900

ctatcc 906

Claims (9)

1. A saline-alkali tolerant Trichoderma strain, which is Trichoderma harzianum (Trichoderma harzianum) M-17, deposited in the chinese type culture collection with the deposit number: CCTCC NO: m2018538.

2. The saline-alkali tolerant trichoderma strain of claim 1, wherein said trichoderma strain is tolerant to salt stress caused by a NaCl concentration of 5% or less; the trichoderma strains are tolerant to alkali stress caused by pH less than or equal to 8.5.

3. The saline alkali tolerant trichoderma strain of claim 1 or 2, wherein said trichoderma strain is tolerant to salt stress and alkali stress caused by a combination of a NaCl concentration of 5% and a pH of 8.5.

4. The method of any one of claims 1-3, wherein Trichoderma harzianum-17 is cultured in the dark at 25-28 ℃ on potato dextrose agar for at least 72h; the potato glucose agar culture medium comprises the following components in parts by weight: 200 parts of potatoes, 1000 parts of distilled water, 15-20 parts of glucose and 8-12 parts of agar powder.

5. The method for culturing the saline-alkali tolerant trichoderma strain according to claim 4, wherein after the trichoderma harzianum-17 is cultured on a potato glucose agar medium for 24 hours, the front surface of a colony is in an aerial hypha white villous shape, and the back surface of the colony is colorless; after the trichoderma harzianum-17 is cultured on a potato glucose agar culture medium for 48 hours, light green conidia are generated on the front surface of a colony, the hyphae are loose and villous, and the back surface of the colony is light green; after the trichoderma harzianum-17 is cultured on a potato glucose agar culture medium for 72 hours, colonies overgrow the whole culture medium, hypha of a middle colony on the front side is white villous, a dark green conidium is generated on the outer ring, and the back of the colony is green.

6. Use of a saline alkali tolerant strain of trichoderma reesei according to any one of claims 1 to 3 for promoting seed germination, seedling growth or/and root growth of a plant.

7. Use of a saline alkali tolerant Trichoderma strain of any one of claims 1 to 3 to alleviate salt stress inhibition during seed germination, seedling growth or/and root growth of a plant.

8. Use according to claim 6 or 7, wherein the plants are alfalfa and rice.

9. A method of enhancing plant growth by a saline alkali tolerant trichoderma strain as claimed in any one of claims 1 to 3, wherein the plant is irrigated with a bacterial suspension comprising a saline alkali tolerant trichoderma strain as claimed in any one of claims 1 to 3; the using amount of the bacterial suspension for irrigating each plant is 50ml, and the content of the trichoderma in the bacterial suspension is (0.9-1.1) multiplied by 10 ⁸ cfu/ml。

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