CN109517743B

CN109517743B - Trichoderma strain ST02 and application thereof

Info

Publication number: CN109517743B
Application number: CN201811629417.9A
Authority: CN
Inventors: 赵忠娟; 扈进冬; 石红蕾; 王玉霞; 李纪顺; 魏艳丽; 董洪芳
Original assignee: Management Center Of Yellow River Delta Sustainable Development Research Institute Of Shandong Province; Ecology Institute Shandong Academy Of Sciences
Current assignee: Management Center Of Yellow River Delta Sustainable Development Research Institute Of Shandong Province; Ecology Institute Shandong Academy Of Sciences
Priority date: 2018-12-28
Filing date: 2018-12-28
Publication date: 2021-09-21
Anticipated expiration: 2038-12-28
Also published as: CN109517743A

Abstract

The invention provides a Trichoderma strain ST02 and application thereof, wherein the Trichoderma strain ST02 is Trichoderma harzianum (Trichoderma harzianum) ST02, and the strain is preserved in the general microbiological center of China Committee for culture Collection of microorganisms at 12-5.2018, with the address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, the preservation number is: CGMCC No. 16964. The trichoderma strain ST02 has strong salt tolerance activity, can promote the salt tolerance of plants, and has the effect of improving saline soil.

Description

Trichoderma strain ST02 and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a trichoderma strain ST02 and application thereof.

Background

The salinized soil in China has wide distribution range and large area, and a large amount of cultivated land has serious salinization, and the improvement and the reasonable utilization of the land resources have important significance for improving the ecological environment and promoting the economic development. At present, bioremediation becomes an effective measure for improving saline-alkali soil by physical methods such as field lifting, salt drainage in a concealed conduit and the like. In recent years, the soil structure is changed by planting salt-tolerant plants, and the reduction of soil salt accumulation becomes a simple and effective biological improvement measure. Researches show that the tolerance of plants to abiotic stress not only depends on genome genetic regulation, but also is related to surrounding complex biological environments, and plant rhizosphere microbial communities can interact with hosts of the plants to form a plant root system, soil and microbial interaction network to support the normal growth of the plants and defend against adverse environments; at present, part of microorganisms are proved to be closely related to the improvement of abiotic stress tolerance of plants, and are applied to saline-alkali soil improvement, and a plant-microorganism combined remediation technology of saline soil is established on the basis, so that a new idea and a new method are provided for biological treatment of soil salinization.

Trichoderma is a ubiquitous, independently living, rapidly propagating fungus, which is well studied for its biological control function. Much of the work on biological control has been done in trichoderma, and recent studies have also found that trichoderma can induce plants to develop tolerance to abiotic stress through trichoderma-plant interrelationships. Researches show that trichoderma strains can promote seed germination and seedling growth of plants under the condition of salt stress, and affect in-vivo penetration protection and Na of the plants⁺/K⁺An ion balance and anti-oxidation system to promote salt tolerance of plants; the bacterial strain can also promote the dissolution of phosphate and the production of phytohormone, and promote the growth of plant seedlings under the condition of salt stress. However, there have been few studies on the tolerance of Trichoderma itself to abiotic stress and its repair ability. Mishra et al system investigated the tolerance of Trichoderma harzianum T103 to both biotic (root pathogens) and abiotic (high salt, heavy metals, pesticides, malathion, carbofuran), and its ability to promote plant growth under stress; singh M and Sharma O P investigated the salt tolerance of 2 Trichoderma longibrachiatum strains, 2 Trichoderma viride strains, and 1 Trichoderma asperellum strains.

The high-efficiency trichoderma harzianum strain for promoting the salt tolerance activity of saline-alkali soil plants is obtained by collecting the trichoderma harzianum with high salt tolerance activity, and researching the influence of the preparation on the salt tolerance of the plants and the improvement of saline soil, and the microbial fertilizer with the salt tolerance characteristic is developed, so that the method has higher theoretical and production significance.

Disclosure of Invention

In view of the above, the invention provides a Trichoderma strain ST02 and application thereof, wherein the Trichoderma strain ST02 is Trichoderma harzianum (Trichoderma harzianum) ST02, is separated from a soil sample collected from Haemonchus estuarinae of yellow river of eastern reclamation, has strong salt tolerance activity, can promote the salt tolerance of plants, and has the effect of improving saline soil.

The technical scheme of the invention is as follows:

trichoderma strain ST02, Trichoderma harzianum ST02, Trichoderma harzianum ST02, which has been deposited in the general microbiological culture Collection center of China Committee for culture Collection of microorganisms, address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, the preservation number is: CGMCC No. 16964.

The trichoderma strain ST02 is obtained by separating from a soil sample collected from the mouth of the eastern reclamation yellow river into the sea, and the soil sample collection site is characterized in that: the salinity of the soil is about 2.18 percent, and the vegetation is less and only tamarix chinensis and suaeda salsa grow.

Trichoderma strain ST02 has all the identifying characteristics of Trichoderma harzianum. The expression is as follows: culturing on a PDA culture medium at 25 ℃, and observing when culturing for 4 days, wherein conidiophores have long main shafts, side branches are always opposite, and phialides are spirally arranged, wherein the width of the main shafts is (1.99-) 2.27-2.88 (-3.25) mu m, the phialides are in a bottle shape, and the length-width ratio of the phialides is (1.65-) 2.38-4.10 (-6.04); observing when the culture is carried out for 7d, wherein spores on the front side of the bacterial colony are yellow-green and hypha are white, the back side of the bacterial colony is orange yellow, and the matrix has orange-yellow water-soluble pigment distribution; the colony surface is in a velvet shape, two circles of yellow-green concentric rings are generated, hyphae are compact, no exudate exists, a large amount of aerial hyphae are generated, and no spore clusters are generated; no obvious smell and no autolysis phenomenon; observed when the conidia are cultured for 14d, the conidia are nearly spherical or oval, the length-width ratio is (1.05-) -1.11-1.48 (-1.74), the conidia are yellow green, and the surface is smooth; observed in 18d culture with intergrown or terminal chlamydospores, which are often oval in shape and spherical in shape.

The trichoderma strain ST02 has stronger salt-tolerant activity, the salt-tolerant rate of the trichoderma strain is 73.9% when treated by 500mM NaCl, and the salt-tolerant rate of the trichoderma strain ST02 can reach 27.9% on a PDA culture medium containing 1000mM NaCl; shake culturing in PDW culture medium containing 0-500mM NaCl for 7d, wherein the hypha growth amount of Trichoderma strain ST02 is higher than control 90%; the hypha growth of the trichoderma strain ST02 was improved by 71% and 67% respectively in PDW medium containing 750mM NaCl and 1000mM NaCl for 7 days.

Application of trichoderma strain ST02 in promoting plant seed germination under salt stress condition.

In the application of promoting the germination of the plant seeds, the application is specifically the application of the trichoderma strain ST02 spore liquid in promoting the germination of the plant seeds under the condition of salt stress.

Preferably, in the above application for promoting plant seed germination, trichoderma strain ST02 spore liquid has a concentration of 2 × (10)⁶-10⁷) CFU/mL; the tomato seed test shows that the using concentration is 2 multiplied by 10⁷Tomato seeds treated with spore liquid of Trichoderma strain ST02 at CFU/mL, under stress of 100mM and 150mM NaCl, showed an increase in seed germination of 103.13% and 591.18%, respectively, compared to controls not treated with strain ST 02.

Application of trichoderma strain ST02 in promoting plant seedling growth under salt stress condition.

In the application of promoting the growth of the plant seedlings, the trichoderma strain ST02 spore liquid is specifically applied to promoting the germination of plant seeds under the condition of salt stress.

Preferably, in the above application for promoting the growth of young plants, the concentration of spore liquid of Trichoderma strain ST02 is 2 × 10⁸CFU/mL; the test using tomato seedlings shows that the tomato seedlings are 2 multiplied by 10⁸The survival rate, the plant height and the fresh weight of the tomato seedlings under the stress of 200mM NaCl are higher than those of the tomato seedlings not treated by the ST02 spore liquid, the survival rate reaches 81.36%, the plant height and the fresh weight of a single plant are the highest and are 127.11% and 158.65% of the comparison respectively, and compared with the seedlings not stressed by NaCl, the reduction rates of the plant height and the fresh weight of the single plant are the lowest and are 4.17 and 7.32% respectively.

The trichoderma strain ST02 can be used for improving the salt-tolerant physiological and biochemical aspects of the plant seedlings under the salt stress condition.

The trichoderma strain ST02 is applied to the aspect of improving the salt-tolerant physiological biochemistry of the plant seedlings, in particular to the application of trichoderma strain ST02 spore liquid to the aspect of improving the salt-tolerant physiological biochemistry of the plant seedlings under the condition of salt stress; the concentration of spore liquid of Trichoderma strain ST02 is 2 × 10⁸CFU/mL; irrigating tomato seedlings by using trichoderma strain ST02 spore liquid under the stress of 200mM NaCl, wherein the chlorophyll content and Pn value of the tomato seedlings are obviously higher than those of a control group which is not treated by the spore liquid; after the tomato seedlings are treated by trichoderma strain ST02 spore liquid, the oxidative damage of salt stress to the tomato seedlings is obviously relieved, the antioxidant enzyme activity of the tomato seedlings is improved, and the inhibition effect of NaCl stress to the antioxidant enzyme activity is relieved; in addition, compared with the control group, the ion leakage rate and the MDA content are obviously lower than those of the control group.

The application of trichoderma strains ST02 in improving saline soil; the application of the trichoderma strain ST02 spore liquid can improve the salinization degree of soil; the Na in soil under the salt stress condition can be reduced by treating with trichoderma strain ST02 spore liquid under the stress of 200mM NaCl⁺And Cl^-Content, increase the number and diversity of microbial communities in soil under salt stress, and increase the total amount of bacteria and fungi.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention provides a trichoderma strain ST02, which has strong salt tolerance activity and can promote plant seed germination, plant seedling growth and plant seedling salt tolerance under the condition of salt stress; in addition, the salinity of the soil can be improved.

2. The tomato seed test shows that the using concentration is 2 multiplied by 10⁷Tomato seeds treated with spore liquid of Trichoderma strain ST02 at CFU/mL, under stress of 100mM and 150mM NaCl, showed an increase in seed germination of 103.13% and 591.18%, respectively, compared to controls not treated with strain ST 02.

3. The test using tomato seedlings shows that the tomato seedlings are 2 multiplied by 10⁸The survival rate, the plant height and the fresh weight of the tomato seedlings irrigated by the CFU/mL spore solution are all higher than those of the tomato seedlings which are not treated by the ST02 spore solution under the stress of 200mM NaCl, and the survival rate reachesTo 81.36%, the plant height and fresh weight of the individual plant were the highest, 127.11% and 158.65% of the control, respectively, and the reduction rates of the plant height and fresh weight of the individual plant were the smallest, 4.17 and 7.32% respectively, compared to the seedlings not stressed with NaCl.

Drawings

In order to make the disclosure of the invention more clear, the following figures are now provided:

FIG. 1 Trichoderma strain ST02 was cultured in PDA for 7d, and observed for front and back morphology and conidium microscopy;

FIG. 2 growth and sporulation of Trichoderma strain ST02 in PDA medium with different concentrations of NaCl;

FIG. 3 salt tolerance of Trichoderma strain ST02 in PDA medium with different concentrations of NaCl;

FIG. 4 hyphal dry weight of Trichoderma strain ST02 in PDW medium with different concentrations of NaCl;

FIG. 5 relative percentage of hyphal dry weight of Trichoderma strain ST02 in PDW medium with varying concentrations of NaCl;

FIG. 6 shows the concentration of 2X 10⁷Germination rate of tomato seeds treated by CFU/mL trichoderma strain ST02 spore liquid under stress of NaCl with different concentrations;

FIG. 7 shows the concentration of 2X 10⁷CFU/mL trichoderma strain ST02 spore liquid treated tomato seeds have root and shoot lengths under stress of different concentrations of NaCl, RL: root length (Root length), SL: shoot length (Shoot length);

FIG. 8 shows 2 × (0, 10)⁵、10⁶、10⁷、10⁸) Germination rate of CFU/mL tomato seeds treated with spore liquid of five Trichoderma strain ST02 with different concentrations under stress of 150mM NaCl;

FIG. 9 shows 2 × (0, 10)⁵、10⁶、10⁷、10⁸) Five trichoderma strains ST02 spore solutions at different concentrations of CFU/mL, applied in different ways (J: seed soaking, G: watering) effect on growth of tomato seedlings under NaCl stress;

FIG. 10 shows 2 × (0, 10)⁵、10⁶、10⁷、10⁸) CFU/mL five trichoderma strains ST02 spore solutions of different concentrations were used to treat the soil, total microbial load in the soil, J: seed soakingG: and (4) watering.

FIG. 11 shows 2X (0, 10)⁵、10⁶、10⁷、10⁸) CFU/mL five trichoderma strain ST02 spore solutions at different concentrations treated soil, total amount of bacteria in soil, J: seed soaking, G: and (4) watering.

FIG. 12 shows 2X (0, 10)⁵、10⁶、10⁷、10⁸) CFU/mL five trichoderma strains ST02 spore liquid with different concentrations are used for treating soil, and the total amount of fungi in the soil is calculated; j: seed soaking, G: and (4) watering.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 isolation of Trichoderma Strain ST02

(1) Preparation of a selective culture medium: the trichoderma strain ST02 is obtained by separating a soil sample collected from the estuary of the yellow river of eastern reclamation by utilizing a selective culture medium of salt-tolerant trichoderma, and the composition of the culture medium is as follows: PDA, 0.4M (mol/L) NaCl, 300mg/L chloramphenicol, 100mg/L streptomycin, 20mg/L rose bengal, and 0.05% Trton X-100; the preparation method of the culture medium comprises the following steps: cooling PDA culture medium containing 0.4mol/L NaCl to below 60 deg.C, adding 300mg/L chloramphenicol, 100mg/L streptomycin, 20mg/L rose bengal and 0.05% Trton X-100, and mixing.

(2) Strain separation: weighing 10g of soil sample, putting the soil sample into a triangular flask added with 90mL of 0.9% NaCl saline, shaking the soil sample for 1h at the temperature of 28 ℃ and at the speed of 160r/min, fully and uniformly mixing the soil sample, taking 1mL of the soil sample, mixing the soil sample into a saline test tube containing 9mL of 0.9% NaCl, vortex, shaking the soil sample for uniform mixing, sequentially carrying out gradient dilution, and taking 1mL of each gradient and adding the gradient into a culture dish with the thickness of 90 mm. Pouring about 20mL of the selective culture medium obtained in the step (1) into each culture dish, slightly rotating and uniformly mixing to uniformly disperse the samples in the culture dish in the culture medium, culturing at 28 ℃ for 3-4 days, and separating and screening strains suspected of trichoderma for identification.

(3) The strains were cultured and observed: the strain is cultured in light and dark for 12h/12h on a PDA culture medium, and the surface morphology of the strain is observed, and the results show that: culturing on PDA culture medium at 25 deg.C for 7 days to obtain white yellow spore mycelium on the front side of the colony, orange yellow spore on the back side of the colony, and orange yellow water soluble pigment distribution in the matrix. The colony surface is in a velvet shape, two circles of yellow-green concentric rings are generated, hyphae are compact, no exudate exists, a large amount of aerial hyphae are generated, and no spore clusters are generated. No obvious smell and no autolysis phenomenon; the shapes of conidiophores, spores and the like are observed by a microscope, and the following results are found: observing at 4d, the conidiophores have long main shafts, the lateral branches are always opposite, and the phialides are spirally arranged, wherein the width of the main shafts is (1.99-) 2.27-2.88 (-3.25) mu m, the phialides are in a bottle shape, and the length-width ratio of the phialides is (1.65-) 2.38-4.10 (-6.04); 14d, the conidia are observed to be nearly spherical or oval, the length-width ratio is (1.05-) -1.11-1.48 (-1.74), the conidia are yellow green, and the surface is smooth; observing at 18d, the chlamydospore with intergrowth or terminal growth is often oval, the terminal growth is often spherical, see figure 1, in figure 1, the first left is the positive colony state of ST02 strain cultured on PDA culture medium for 7 days, the second left is the negative colony state of ST02 strain cultured on PDA culture medium for 7 days, and the third left is the conidiophore form observed under microscope after ST02 strain cultured on PDA culture medium for 7 days.

(4) And (3) strain identification: the genomic DNA of Trichoderma was extracted using the Fungi DNA Mini Kit (Omega, USA), amplified using primers ITS1-F/ITS4, and the PCR product was sequenced by crossing to Producer Biolabs GmbH, where NCBI and ISTH were aligned to find that the strain ST02 was Trichoderma harzianum (Trichoderma harzianum) designated Trichoderma harzianum (Trichoderma harzianum) ST 02.

EXAMPLE 2 Trichoderma Strain ST02 determination of salt tolerance Activity

The method comprises the following steps: in order to examine the growth and sporulation of Trichoderma strain ST02 under stress of different salt concentrations, it was inoculated onto PDA solid medium containing different concentrations of NaCl (0, 100, 250, 500, 750, 1000mM), and the colony radius and sporulation were observed and measured at 5d growth.

In order to quantitatively measure the influence of NaCl on the growth of trichoderma strains, the selected trichoderma strains ST02 were inoculated onto PDW liquid culture media containing NaCl (0, 100, 250, 500, 750, 1000mM) at different concentrations, shake-cultured for 7d at 25 ℃, and mycelia were collected, dried, and weighed and relative percentages of dry weight of mycelia were measured.

As a result: the growth speed of trichoderma strain ST02 bacterial colony is reduced along with the increase of NaCl concentration, the growth is faster on the PDA culture medium without NaCl, the whole culture dish is basically covered, and a large amount of spores are generated; 100mM and 250mM NaCl had less effect on Trichoderma growth and faster growth but inhibited spore production, see FIG. 2; under the stress of 500mM NaCl, the salt tolerance of trichoderma strain ST02 reaches 73.9%; the salt tolerance of trichoderma strain ST02 can still reach 27.9% on PDA culture medium containing 1000mM NaCl.

The trichoderma strain ST102 is subjected to shake culture in PDW containing NaCl of different concentrations for 7d, the dry weight of hyphae is in a decreasing trend along with the increase of the NaCl concentration, the change of the dry weight of the hyphae is not obvious when the hyphae is treated by NaCl of less than 500mM, the dry weight of the hyphae is more than 90 percent compared with a control, the dry weight of the hyphae is obviously reduced when the hyphae is treated by NaCl of 750mM and 1000mM, and the dry weight of the hyphae is 71 percent and 67 percent of the control respectively compared with the control, as shown in the graph 4-5.

Example 3 application of Trichoderma strain ST02 in germination from plant seeds under salt stress conditions

The method comprises the following steps: the sterilized filter paper was placed in a sterilized petri dish, moistened with NaCl solutions of different concentrations (0, 50, 100, 150, 200mM), and wetted with 2X 10⁷Soaking tomato seeds in CFU/mL trichoderma strain ST02 spore solution for 3-4h, uniformly placing the treated seeds on wetted filter paper, repeating the treatment for 3 times for 60 seeds in each dish, culturing in an incubator at 25 ℃, counting the germination rate of the seeds, and measuring the root length and the bud length of the seeds after 7 days of germination.

To determine the optimal concentration for Trichoderma use, sterilized filter paper was placed in a sterilized petri dish, moistened with a 0, 150mM NaCl solution, and treated with different concentrations of 2 × (0, 10)⁵、10⁶、10⁷、10⁸) Soaking tomato seeds for 3-4h by using CFU/mL trichoderma strain ST02 bacterial suspension, and uniformly placing the treated seeds60 granules are placed in each dish on moistened filter paper, each treatment is repeated for 3 times, the culture is carried out in an incubator at 25 ℃, the germination rate of the seeds is counted, and the optimal spore liquid concentration of trichoderma strain ST02 required for treating the tomato seeds is screened.

As a result: by 2X 10⁷The germination rate detection results of tomato seeds treated by CFU/mL trichoderma strain ST02 spore liquid under the stress of NaCl with different concentrations show that under the stress conditions of 0 and 50mM NaCl, the strain ST02 has no obvious influence on the germination rate of the tomato seeds, and under the stress conditions of 100mM and 150mM NaCl, the trichoderma strain ST02 significantly increases the germination rate of the tomato seeds by 103.13% and 591.18% respectively compared with a control which is not treated by the trichoderma strain ST 02; tomato seeds treated and untreated with trichoderma strain ST02 under 200mM NaCl stress were difficult to germinate, see fig. 6-7; under the condition of 0mM NaCl, the trichoderma obviously increases the length of the root length and has no obvious influence on the bud length; in 50mM NaCl treatment, the control group has no obvious difference between the root length and the bud length compared with the control group without NaCl treatment, and the application of Trichoderma strain ST02 has obvious longer root length and obviously shorter bud length compared with the application of Trichoderma strain ST 02; under the condition of 100mM NaCl treatment, the root length and the bud length are obviously shortened compared with those without NaCl, and the elongation of the root and the bud is obviously promoted by applying Trichoderma strain ST02 treatment; under 150mM NaCl treatment, the bud did not grow, and the application of Trichoderma strain ST02 significantly promoted root elongation; the results show that trichoderma promotes the elongation of the roots and buds of the tomato seeds after germination under NaCl stress, and are shown in fig. 6-7.

The screening result of the optimal use concentration of the trichoderma spore liquid shows that: 2X 10⁷And 2X 10⁶Tomato seeds soaked in spore liquid of trichoderma strain ST02 at CFU/mL concentration showed the highest germination rates under 150mm nacl stress, 25% and 26.7%, respectively, which were increased 373.13% and 398.51% over the control, see fig. 8.

The results show that the salt-tolerant trichoderma strain ST02 can promote the germination of tomato seeds under salt stress, and the optimal trichoderma spore liquid use concentration is 2 x (10)⁶-10⁷)CFU/mL。

Example 4 application of Trichoderma strain ST02 to promotion of plant seedling growth under salt stress conditions

Taking soil from a field without trichoderma liquid, adding the soil into a paper cup, sowing tomato seeds with 130g of soil, performing trichoderma treatment and NaCl stress treatment, and setting a control group respectively; after NaCl treatment for 2 weeks, the survival rate, plant height and fresh weight of tomato seedlings were tested.

The pot experiment was specifically designed as follows:

(1) trichoderma strain ST02 application method: the spore liquid of trichoderma strain ST02 is applied by two modes of seed soaking and pouring;

seed soaking treatment: trichoderma strain ST02 spore liquid was resuspended and diluted to different concentrations 2 × (0, 10)⁵、10⁶、10⁷、10⁸) CFU/mL, soaking tomato seeds for 3-4h by using the resuspended spore solution, sowing the treated tomato seeds into prepared nutrient soil, wherein 15 tomato seeds are planted in each pot, and 6 tomato seeds are planted in each pot and divided into two groups for comparison and NaCl treatment respectively; meanwhile, tomato seeds which are not treated by ST02 spore liquid are used as blank control, and the same number of untreated seeds are sowed in parallel and divided into two groups of control and NaCl treatment for irrigating trichoderma spore liquid;

and (3) irrigation treatment: germinating un-soaked tomato seed, growing 2 true leaves, diluting to different concentrations 2 × (10)⁵、10⁶、10⁷、10⁸) Irrigating with 6 pots of CFU/mL trichoderma spore solution, dividing into two groups for control and NaCl treatment;

(2) NaCl stress treatment: after trichoderma strain ST02 spore liquid treatment, when tomato seedlings grow to 5-6 true leaves, treating the tomato seedlings with 200mM NaCl for 1 time 2-3 d for 3 times, and after NaCl treatment for 2 weeks, determining the survival rate, plant height and fresh weight of the seedlings.

As a result: the growth conditions of the tomato seedlings after different treatments are observed and found as follows: using 2X 10⁸Tomato seedlings irrigated with CFU/mL Trichoderma strain ST02 spore liquid grew best, were strongest without NaCl treatment, and had the highest survival rate with 200mM NaCl treatment, as shown in FIG. 9; statistics of the survival rate, plant height and fresh weight of the seedlings of the different treated tomatoes show that the survival rate of the tomato seedlings of the control group which is not treated with the spore liquid of trichoderma strain ST02 under the treatment of 200mM NaClAbout 40.83%, the survival rate of the tomato seedlings treated with the seed soaking was slightly higher than that of the control group except for the use of 2X 10⁶CFU/mL spore fluid-inoculated tomato seedlings (no significant change compared to control); tomato seedlings irrigated with different concentrations of spore solutions showed a large difference of 2X 10 under 200mM NaCl treatment⁸The survival rate of tomato seedlings irrigated by the CFU/mL trichoderma spore solution reaches 81.36%, 2 multiplied by 10⁵The survival rate of the tomato seedlings irrigated by the CFU/mL trichoderma spore solution is similar to that of the soaked tomato seedlings, and is 2 multiplied by 10⁶And 2X 10⁷The survival rate of CFU/mL spore solution irrigation was slightly lower than that of the control group, which is shown in Table 1.

The plant height statistics result shows that the tomato seedlings are not stressed by NaCl, and 2 multiplied by 10⁷And 2X 10⁸The plant height of the CFU/mL spore solution after seed soaking treatment is slightly higher than that of the control group, 2 multiplied by 10⁵And 2X 10⁶The plant height after the CFU/mL spore solution seed soaking treatment is not obviously different from that of a control group, the plant height after the spore solution with different concentrations is irrigated is obviously higher than that of the control group, and the plant height is 2 multiplied by 10⁸The plant height is highest when CFU/mL spore solution is used for irrigation treatment; the plant heights of the control group and each treatment group are reduced to different degrees under 200mM NaCl treatment, and the plant heights of the trichoderma spore liquid-treated groups are higher than those of the control group, wherein the plant heights are 2 multiplied by 10⁸The height of the tomato seedlings irrigated by CFU/mL spore solution is the highest, and the reduction rate of the height of the tomato seedlings is the smallest compared with the seedlings without NaCl stress, is 4.17 percent and is obviously lower than 14.72 percent of the control group, and the table 1 shows.

The fresh weight of individual seedlings showed that, when not treated with NaCl, the fresh weights of individual tomato seedlings treated with different concentrations of Trichoderma strain ST02 spore liquid were all higher than the control group, and the fresh weights of individual irrigation-treated tomato seedlings were higher than the seed soaking treatment, wherein 2X 108C^FThe fresh weight of the tomato seedlings irrigated by the U/mL spore solution is the highest and is 53.02% higher than that of the control group; under 200mM NaCl treatment, the fresh weight of the control group and each treatment individual plant is obviously reduced, wherein the fresh weight is 2 multiplied by 10⁸The fresh weight of each plant treated by pouring CFU/mL spore solution is the highest, and compared with a control not treated by NaCl, the fresh weight of each plant is 2 multiplied by 10⁸The reduction rates of fresh weight of individual plants were similar at CFU/mL seed soaking and watering, 7.45% and 7.32%, respectively, as shown in Table 1.

As described above, TrichodermaThe strain ST02 spore liquid treatment can promote the growth of tomato seedlings under NaCl stress, the survival rate, the plant height and the fresh weight of the seedlings under 200mM NaCl stress are higher than those of the tomato seedlings not treated by the ST02 spore liquid, and the concentration is 2 multiplied by 10⁸The promotion effect of CFU/mL spore liquid irrigation on tomato seedlings is optimal, and the survival rate, the plant height and the fresh weight of a single plant are the highest.

TABLE 1 survival rate, plant height and fresh weight of seedlings treated differently

J: seed soaking, G: irrigation

Example 5 application of Trichoderma strain ST02 in salt stress to promote salt tolerance of plants

Soil is taken from a field without trichoderma liquid application, the soil is added into a paper cup, each soil is about 130g, tomato seeds are sown, trichoderma strain ST02 spore liquid treatment and NaCl stress treatment are carried out, and a control group is respectively set. After NaCl treatment for 2 weeks, detecting salt-tolerant physiological and biochemical reactions of the tomato seedlings, comprising: chlorophyll content, net photosynthetic rate (Pn value), ion leakage rate, MDA content, and antioxidase (SOD, POD, CAT) activity.

(1) A potting test was performed using the potting test design provided in example 4;

(2) chlorophyll content and Pn value determination: taking 2 full-spread leaves at the top ends of the tomato seedlings after different treatments respectively, and measuring the Pn value by using a portable LI6400(LI-COR, USA) photosynthetic rate tester; LI6400 leaf cell 6400-15 (diameter 1.0cm) with illumination intensity of 250. mu. mol photons. m^-2·s^-1The internal concentration of the saturated light is 500mg/m³CO₂Injecting into system, and maintaining relative humidity at 60-70% and leaf surface temperature at 22-25 deg.C during measurement;

a method for detecting chlorophyll content of leaves refers to Zhangliang and the like, wherein the leaves after different treatments are taken, thick veins are cut and cut into fragments, 0.5g of the leaves are weighed and ground into homogenate by 5mL of acetone, the supernatant after centrifugation is subjected to constant volume of 20mL by 80% of acetone, 1mL of pigment extracting solution is taken, 4mL of 80% of acetone is added for dilution, optical density values at 663nm and 645nm are measured by a spectrophotometer, and 80% of acetone is taken as a reference. The chlorophyll content was calculated according to the following formula:

Ca(mg/L)＝12.7OD₆₆₃-2.69OD₆₄₅

Cb(mg/L)＝22.9OD₆₄₅-4.68OD₆₆₃

Ct(mg/L)＝8.02OD₆₆₃+20.21OD₆₄₅

chlorophyll content (mg/g leaf) ═ Ct (mg/L) × extract volume (L) × dilution factor/fresh leaf weight (g)

(3) And (3) detecting the relative conductivity and the MDA content: the method comprises the steps of using leaves of tomato seedlings treated differently as materials, measuring the relative conductivity and MDA content of the leaves of the seedlings treated by the spore liquid of the Trichoderma strain ST02 and not treated by the spore liquid under the NaCl stress condition, and measuring the relative conductivity and MDA content of the leaves by referring to Lv and the like.

And (3) measuring the relative conductivity: selecting leaves of treated seedlings, shearing thick veins into fragments of 0.5 multiplied by 0.5cm, randomly selecting 20 fragments for each treatment, soaking in deionized water for 24h, measuring the conductivity of the soaked solution, boiling the soaked solution of the leaves in boiling water for 15min, rapidly cooling, and measuring the conductivity; the calculation formula of the relative conductivity is as follows:

relative conductivity ═ S1-C1)/(S2-C2) × 100%

S1: conductivity of sample before cooking, C1: blank conductivity of water before cooking, S2: conductivity of the sample after cooking, C2: blank conductivity of water after cooking.

And (3) MDA content determination: selecting 0.2g of leaves, grinding the leaves in 5mL of 10% trichloroacetic acid, centrifuging the leaves at 12000rpm for 10min, mixing 2mL of supernate with 2mL of 0.6% thiobarbituric acid, reacting the mixture in a boiling water bath for 15min, rapidly cooling the mixture and centrifuging the mixture; the light absorption of the supernatant was measured at wavelengths of 532nm, 600nm and 450nm, and the concentration of malondialdehyde was calculated according to the following formula: c (. mu. mol. L-)¹)＝6.45×(OD₅₃₂-OD₆₀₀)-0.56×OD₄₅₀The MDA content is expressed in μmol g^-1FW (fresh weight) as follows

(4) Determination of antioxidant enzyme activity: taking 0.2g of leaves of tomato seedlings treated differently, placing in a precooled mortar, adding 1mL of precooled 50mM potassium phosphate buffer (pH 7.8), grinding in an ice bath to homogenate, adding buffer to wash until the final volume is 5mL, pouring into a 7mL centrifuge tube, centrifuging at 4 ℃ and 12000rpm for 20min, and respectively using photochemical reduction method of Nitrotetrazolium (NBT), guaiacol method, H₂O₂The activities of superoxide dismutase (SOD), Peroxidase (POD) and Catalase (CAT) were measured.

(5) As a result: when not treated with NaCl, the chlorophyll content and Pn value of the Trichoderma strain ST02 spore liquid and the tomato seedlings without spore liquid are not obviously different; the chlorophyll content and Pn value are obviously reduced when 200mM NaCl is treated, and the reduction degree of spore liquid of Trichoderma strain ST02 is reduced, so that the chlorophyll content and Pn value of tomato seedlings after spore liquid of Trichoderma strain ST02 is obviously higher than that of a control, and the concentration of the chlorophyll and Pn value is 2 × 10⁸The chlorophyll content and Pn value of tomato seedlings irrigated by CFU/mL trichoderma strain ST02 spore liquid are the highest, and are shown in Table 2; the influence of the spore liquid on the particle leakage rate and the MDA content of the tomato seedlings without NaCl stress is small, when the tomato seedlings are treated by 200mM NaCl, the ion leakage rate and the MDA content of the tomato seedlings are obviously increased, the increase amplitude is reduced after the spore liquid is applied, and the increase amplitude is also 2 multiplied by 10⁸The ion leakage rate and MDA content of tomato seedlings irrigated by CFU/mL spore liquid are lowest, and the ion leakage rate and the MDA content are shown in a table 2. The NaCl-unstressed tomato seedlings, to which spore liquid was applied to increase the antioxidase activity of the tomato seedlings, also showed 2X 10⁸Tomato seedlings irrigated with CFU/mL spore solution had the most increased antioxidant activity, and 200mM NaCl stressed reduced antioxidant activity, which was similarly shown to be 2X 10⁸CFU/mL, spore fluid-watered tomato seedlings showed the lowest reduction in antioxidant enzyme activity, showing the highest levels, see Table 2. Description of the use of 2X 10⁸The tomato seedlings irrigated by the CFU/mL trichoderma spore liquid have the strongest salt tolerance.

TABLE 2 determination of physiological and biochemical indexes for salt tolerance of seedlings treated differently

J: seed soaking, G: irrigation

Example 6 application of Trichoderma strain ST02 in improving saline soil

The method comprises the following steps: the pot test was designed as described in example 4. After NaCl treatment for 2 weeks, collecting rhizosphere soil of tomato seedlings treated with 200mM NaCl, applied with Trichoderma strain ST02 spore liquid and not applied with spore liquid, and detecting Na in the soil⁺、Cl^-And the change of the number of microorganisms in the soil.

Na⁺、Cl^-The determination method comprises the following steps: 1) detection of Cl by silver nitrate titration^-Measuring Na content by flame photometry⁺Content (c); 2) detection of Na by atomic absorption spectrophotometer⁺Content, determination of Cl by ion chromatography^-And (4) content.

The method for detecting the quantity of the microorganisms in the soil comprises the following steps: extracting soil genome DNA by using kit, and using Femto^TMBacterial/Fungal DNA Kit was used to perform PCR to detect the approximate number of bacteria and fungi in the soil.

As a result: for Na in soil⁺And Cl^-The content detection result shows that Na in the soil is not treated by NaCl⁺And Cl^-The content is low, and Na in the soil is obtained after the spore liquid is applied⁺And Cl^-The content was slightly decreased. Na in soil when treated with 200mM NaCl⁺And Cl^-Obviously increases Na in soil applied with spore liquid⁺And Cl^-The content of the Na in the soil is obviously lower than that of the non-applied control group soil⁺And Cl^-Content and utilization concentration of 2X 10⁸Na in CFU/mL spore liquid irrigated soil⁺And Cl^-The content is lowest, see table 3; the total amount of microorganisms in the soil is obviously increased by applying the spore solution, the total amount of bacteria in the soil is also slightly increased, the total amount of fungi in the soil is obviously increased, the total amount of the microorganisms in the soil is obviously reduced in comparison with the soil without NaCl stress under the stress of 200mM NaCl, and the total amount of the microorganisms, the total amount of the bacteria and the true bacteria under the stress of NaCl is relieved by applying the spore solutionThe effect of total bacteria showed that the total microbial, bacterial and fungal counts in trichoderma soil applied under 200mm nacl stress were significantly higher than the control, see fig. 10-12.

In conclusion, application of Trichoderma can reduce Na in soil under salt stress conditions⁺And Cl^-The content, the number and the diversity of microbial communities in the soil under salt stress are increased, and the salinization degree of the soil is improved.

TABLE 3 Na in different treated soils⁺And Cl^-Content (wt.)

J: seed soaking, G: irrigation

Therefore, the trichoderma strain ST02 provided by the invention has stronger salt-tolerant activity, and can promote plant seed germination, plant seedling growth and plant seedling salt tolerance under the condition of salt stress; in addition, the salinity of the soil can be improved.

Claims

1. Trichoderma strain ST02, Trichoderma harzianum ST02, Trichoderma harzianum ST02, which has been deposited in the general microbiological culture Collection center of China Committee for culture Collection of microorganisms, address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, the preservation number is: CGMCC No. 16964.

2. Use of the trichoderma strain ST02 according to claim 1 for promoting germination of tomato seeds under salt stress conditions.

3. The use of trichoderma strain ST02 for promoting the germination of tomato seeds under salt stress conditions as claimed in claim 2, in particular the use of trichoderma strain ST02 sporophore to promote the germination of tomato seeds under salt stress conditions.

4. Trichoderma strain ST02 according to claim 3 under salt stress conditionsThe application of promoting tomato seed germination is characterized in that the concentration of trichoderma strain ST02 spore liquid is 2 x (10)⁶-10⁷)CFU/mL。

5. Use of the Trichoderma strain ST02 of claim 1 for promoting the growth of tomato seedlings under salt stress conditions.

6. The use of Trichoderma strain ST02 in promoting tomato seedling growth under salt stress conditions according to claim 5, in particular Trichoderma strain ST02 sporophore in promoting tomato seedling growth under salt stress conditions.

7. Use of Trichoderma strain ST02 in promoting tomato seedling growth under salt stress conditions according to claim 6, wherein Trichoderma strain ST02 sporophore fluid concentration is 2 x 10⁸CFU/mL。

8. The use of Trichoderma strain ST02 according to claim 1 for improving salt tolerance physiological and biochemical aspects of tomato seedlings under salt stress conditions.

9. The application of trichoderma strain ST02 in improving the salt tolerance physiology and biochemistry of tomato seedlings according to claim 8 is characterized in that the trichoderma strain ST02 spore liquid is applied in improving the salt tolerance physiology and biochemistry of tomato seedlings under the condition of salt stress.

10. Use of Trichoderma strain ST02 according to claim 1 for improving saline soil.