CN109112072B - Application of trichoderma in promoting plant growth - Google Patents

Abstract

The invention discloses application of trichoderma in promoting plant growth. The Trichoderma of the invention is Trichoderma asperellum (Trichoderma asperellum) and has the strain number: HTTA-Z0002 with the preservation number of CGMCC No. 15677. The HTTA-Z0002 strain can degrade phosphorus, potassium and cellulose, produce phytohormone, siderophores and the like, promote plant growth, induce plant stress resistance and salt resistance, has a multifunctional promotion effect on plant growth, and can reduce the use of fertilizers.

Description

Application of trichoderma in promoting plant growth

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to application of trichoderma in promoting plant growth.

Background

The agricultural production can not be separated from fertilizers, the abuse of chemical fertilizers increases the production cost, soil hardening, organic matter scarcity, decrease of farmland fertility, large-area nitrogen, phosphorus and potassium deficiency, the quality of agricultural products is reduced, soil and environment are polluted more seriously, and secondary salinization of the soil is heavier and heavier, so that the development of microbial fertilizers is a strategic development plan for solving the agricultural fertilizers and protecting the soil and the environment in China at present.

Trichoderma becomes the most concerned fertilizer probiotics due to rich enzymes, plant growth promotion effect and environment safety, the functions and effects of the viable bacteria on plant growth are directly influenced by different habitats, compared with terrestrial habitat trichoderma, marine habitat trichoderma is less in development and research and belongs to a new habitat microorganism, and as the marine habitat is worse than terrestrial, trichoderma living in a special habitat has lower nutritional requirements and lower artificial culture cost, the plant promotion effect is diversified, and the good development and application prospect is displayed.

The existing saline-alkali soil with the area of more than 0.2 hundred million hm2 in cultivated land of China has a tendency of increasing year by year, because of the national situation of more people and less land, the improvement of the productivity of saline soil is always advocated by the nation to local governments, microbial fertilizer is metabolized near plants to play a role, most of the microbial fertilizer is live bacterial preparation, the microbes from land cannot normally play a role because the saline soil has higher salt content, the growth of the microbes is influenced, oceans especially the unique geographical position of the polar region and extreme environments of extremely cold, high salt and the like cause various adaptive lives of the microbes, the microbes can better play a role in the saline land, and simultaneously, the generated functions on the plants are more than those on the land.

The microbial fertilizer mainly plays a role in generating enzyme and beneficial substances by microbial life activity, phosphorus and potassium are major elements necessary for plant growth, a large amount of inorganic phosphorus and potassium in the form of silicate minerals exist in soil, the soil is difficult to absorb by plants due to poor solubility, the enzyme and the beneficial substances generated by some microbes can convert insoluble phosphorus and potassium elements into soluble states for the plants to utilize, and the microbes capable of generating phytase can also convert phytic acid which cannot be utilized by the plants into effective phosphorus elements which can be utilized by the plants; indoleacetic acid (IAA) is a plant endogenous auxin, participates in regulation and control of various physiological and biochemical processes such as cell growth, cambium division and the like, cannot be lacked in plant growth, and microorganisms around plants can also have a good complementary effect on the plants when producing IAA; most of iron elements in the nature exist in an insoluble state and are difficult to utilize by plants, an iron carrier is a small molecular compound with strong specific chelation of Fe3+, and a plurality of plant rhizosphere probiotic microorganisms can absorb iron in the environment by synthesizing the substance, compete for the iron with pathogenic bacteria and provide redundant iron for the plants to utilize. The iron carrier can also chelate metal ions such as manganese, lead, mercury, chromium, cadmium and the like, and has important significance in the fields of soil environment restoration, food safety and the like.

Cellulose is the most widely distributed resource on the earth, but other organisms including plants cannot be directly utilized, only crop wastes in China can reach 0.7 multiplied by 109 tons every year, most of the raw materials are incinerated, the utilization rate is low, and great pressure is brought to environmental protection. The cellulose degradation efficiency by using the cellulase produced by beneficial microorganisms is high, the pollution is avoided, and the double economic and ecological values are realized. The cellulase is a complex enzyme system consisting of endoglucanase, cellobiohydrolase and the like, and the Eg1 gene is a key gene for synthesizing the cellulase by microorganisms, and the sensitivity and the accuracy of the Eg1 gene become effective indexes for people to evaluate whether strains produce the cellulase or not.

In order to be better utilized by human beings and industrially produced, the strain meets the condition that the fertilizer and the production fermentation characters reach the standard, after the microbial strains are obtained through various screening models, mutation breeding is usually carried out, if the operation is successful, the economic value can be improved by hundreds of or even thousands of times on the basis of wild strains, the liquid fermentation of the microorganisms is the basis of embodying low cost and easy industrial production, but the spore yield of trichoderma in liquid culture is less, the solid culture restricts large-scale production, and the viable bacteria preparation of the trichoderma strains is conidia, namely the quantity of the conidia is in direct proportion to the fertilizer efficiency. Aiming at the most important bacteriostatic activity and liquid culture sporulation yield characteristics of the trichoderma fertilizer strain, the strain HTTA-Z0002 obtained by mutation breeding can promote plant growth and stress resistance, inhibit soil pathogenic bacteria and also has good characteristics of artificial fermentation culture production.

Disclosure of Invention

The invention aims to provide application of trichoderma in promoting plant growth.

The Trichoderma asperellum (Trichoderma asperellum) is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2018, 5 months and 17 days, and the strain number is as follows: HTTA-Z0002 with the preservation number of CGMCC No. 15677; xilu No. 3, Beijing Chaoyang district, Beichen, China.

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

use of a Trichoderma fungus, Trichoderma asperellum (Trichoderma asperellum), strain No.: HTTA-Z0002 with the preservation number of CGMCC No. 15677.

Further, the use comprises degrading phosphorus, potassium, calcium phytate or cellulose.

Further, the application comprises promoting the germination of plant seeds and the growth of seedlings.

A fertilizer comprising the above trichoderma.

According to the invention, on two culture media for conventionally testing phosphorus and potassium, the HTTA-Z0002 trichoderma strains have obvious hydrolysis rings, and the strains have good phosphorus and potassium degradation effects; the hydrolysis ring is also obvious on the culture medium containing calcium phytate, namely the calcium phytate can be hydrolyzed, which shows that the phytase producing capability is realized, and the phytic acid which can not be utilized by plants can be converted into the available phosphorus element which can be utilized by the plants; the strain can grow on a culture medium with cellulose as a unique carbon source, and simultaneously, an endoglucanase gene Eg1 which is a key gene for synthesizing cellulase by the strain is amplified by PCR (polymerase chain reaction), so that the strain has the capacity of degrading cellulose; the fermentation liquor of the trichoderma is subjected to Salkowski colorimetric solution color development detection, the result shows red, indoleacetic acid is also detected by adopting an HPLC method, the strains can generate IAA, and the yield of the indoleacetic acid of the trichoderma HTTA-Z0002 strain obtained by the internal standard method is 2.09 mg/L; the strain can make the CAS detection culture medium turn purple red, which shows that the strain has the capability of producing siderophores, the relative content of the siderophores increases along with the culture time, and reaches a maximum value of 83.9 percent at the 7 th day.

The HTTA-Z0002 strain has a promoting effect on the germination of plant seeds and the growth of seedlings, tomato plant seedlings treated by the strain liquid are stronger than seedlings treated by clear water control, the leaves are large, the leaf color is dark green, the stems are thick, the root hairs are long, and the average plant height of the seedlings is increased by 70%; the root length is increased by 96.6 percent; the stem thickness is increased by 85.7 percent, the chlorophyll a, the chlorophyll b and the total chlorophyll of the tomato leaves are respectively increased by 50.0 to 125.0 percent compared with the control, and the average fresh weight and dry weight are respectively increased by 86.0 to 128.6 percent

When the strain is cultivated in saline soil, the HTTA-Z0002 strain has a large promotion effect on tomato seed germination, radicles and hypocotyls are both cultured in the soil close to normal, the germination index is obviously higher than that of a control, and the germination index is improved by 56.7%. The contents of enzymes SOD, POD, CAT, PPO, PAL, proline, soluble sugar and the like related to stress resistance of the plant are obviously improved, the improvement range is 39.7-87.5%, and the difference is obvious compared with CK (P is less than 0.05). After being treated by HTTA-Z0002 bacteria, the Na + content in the roots and the leaves of the tomatoes is reduced to different degrees, and compared with a control, the Na + accumulation in the roots and the leaves is reduced by 43.1 to 65.2 percent, the K + content in the roots and the leaves is increased, and the K + content in the roots and the leaves is increased by 37.0 to 46.8 percent.

The application effect of the invention is that: the HTTA-Z0002 strain can degrade phosphorus, potassium and cellulose, produce phytohormone, siderophores and the like, promote plant growth in various aspects, induce plant stress resistance and salt resistance, has a multifunctional promotion effect on plant growth, and can reduce the use of fertilizers. The artificial culture fermentation production is easy to operate, low in cost and safe to the environment.

Drawings

FIG. 1 is a graph of related branching trees.

FIG. 2 detection of IAA absorption peak by HPLC

Detailed Description

The following specific examples are further illustrative of the methods and techniques provided by the present invention and should not be construed as limiting the invention thereto.

Example 1: classification and identification of trichoderma strains

Trichoderma asperellum HTTA-Z0002 strain in solid MPDA culture medium (potato extract 200 ml, peptone 1-2g, glucose 25-30g, NaCl 5-8g, MgCl₂6H₂0.1-0.3g of O, 0.1g of KCl and 1000ml of agar 18g of water), culturing for 3-5 days at 20-23 ℃, continuously observing and recording changes of colony morphology, color and the like, and selecting hyphae to observe sporulation structure under an optical microscope. The strain grows vigorously on an MPDA artificial culture medium, a spore-forming layer and a conidiophor are dense like a felt shape, a green color or a white color, the conidiophor has a diaphragm, lateral branches are generated, the branches are vertically opposite, the base is wider, the tail end is thinner and is in a bottle shape, a conidiophor is single-spore, spherical to oval shape and colorless, and a more obvious conidiophor group is formed at the tip.

The strains were cultured as described above, sequenced by PCR amplification products using universal primers for fungal ITS genes by conventional methods, and compared by the BLAST program of NCBI, sequence homology was analyzed and affinity tree-branching was made to compare closely related species (see FIG. 1). The strain is identified to be Trichoderma asperellum by combining the biological colony morphology, color and microscopic structure of spore-forming structure and molecular biological information of the strain and referring to relevant Trichoderma classification and identification monographs at home and abroad.

Example 2: mutation breeding of trichoderma strains

A trichoderma strain screened by multiple fertilizer activity tests is subjected to conventional ultraviolet-microwave composite mutagenesis, a monoclonal colony which grows vigorously is selected, an MPDA culture medium, a yeast sucrose (ESA) culture medium and a Richard culture medium which are commonly used for trichoderma are firstly adopted to test the growth and spore production conditions, an MPDA culture medium which has better growth and spore production prevalence is selected, the strain with abnormal growth is cultured for 5 days in a solid MPDA culture medium, then liquid MPDA is inoculated, the strain is cultured for 7 days at 20 ℃, 23 and 25 ℃, the spore production quantity is counted and compared in a microscopic mode, the strain with the spore production quantity exceeding 50 percent is selected, the selected strain is continuously cultured for 5 generations in a liquid mode, the growth and spore production stability of the strain is observed by adopting the same spore production quantity test method, and the finally selected HTTA-Z0002 trichoderma strain has the maximum spore production quantity of 4.25 multiplied by 10⁸cfu/cm²Compared with wild bacteria, the yield is improved by 150.7 percent.

Example 3: laboratory detection of plant effects

HTTA-Z0002 strain was cultured using a special test medium reported and tested by observation and calculation: respectively measuring the diameter (D/mm) of the bacterial colony and the diameter (D/mm) of the hydrolysis ring by a cross multiplication method according to the sizes of the bacterial colony and the hydrolysis ring, calculating the D/D value, and detecting the capacities of dissolving phosphorus and potassium, degrading cellulose and producing phytase; measuring the indole acetic acid producing capacity by adopting a conventional HPLC method, and calculating the indole acetic acid yield; the siderophore production ability of trichoderma was examined by reported semi-PDA and CAS media, and the relative siderophore content (%) of [ (Ar-As)/Ar ] × 100% was calculated using the absorbance value thereof As a reference value (Ar). Meanwhile, the primer XWS-F/XWS-R is used for PCR amplification to verify whether the gene has the endoglucanase Eg1 degrading gene or not, and the obtained Eg1 gene is subjected to sequence comparison analysis.

On two culture media for testing phosphorus and potassium, a hydrolysis ring around a bacterial colony of the HTTA-Z0002 trichoderma strain is obvious, which indicates that the strain has stronger phosphorus and potassium dissolving capacity.

TABLE 1 Trichoderma degrading P-K and producing phytase and cellulase

Function of	Diameter of hydrolysis ring (D) (mm)	Colony diameter (d) (mm)	D/d
				Degradation of phosphorus	40.4±0.3	24.8±0.4	1.63
Degrading potassium	45.1±0.4	28.2±0.3	1.59
				Phytase production	40.0±0.2	17.2±0.3	2.37
Production of cellulase	71.2±0.5	46.1±0.4	1.76

On a culture medium containing calcium phytate, an obvious hydrolysis ring is generated around trichoderma harzianum colonies, namely, calcium phytate can be hydrolyzed, and the phytase production capability is shown; the HTTA-Z0002 strain can make the CAS detection culture medium turn purple red, which shows that the CAS detection culture medium has the capability of producing siderophores, and the relative content of the siderophores increases along with the culture time, reaches the maximum value at the 7 th day and is 83.9 percent.

The fermentation liquor of the trichoderma is subjected to Salkowski colorimetric solution color development detection, and the result shows that the trichoderma is red, which indicates that the strain can generate IAA; and (3) detecting a fermentation liquid sample by HPLC (high performance liquid chromatography), wherein the sample has an absorption peak at the 18 th min position, calculating by calculus to obtain a peak area, and obtaining the yield of trichoderma indole acetic acid to be 2.09mg/L by combining an indole acetic acid standard curve and an internal standard method.

The HTTA-Z0002 strain can grow on a culture medium with cellulose as a unique carbon source, which shows that the HTTA-Z0002 strain can utilize cellulolytic as the carbon source, namely has the capacity of degrading fibers, a specific band is about 750bp by utilizing primer XWS-F/XWS-R PCR amplification, the homology of the specific band and Trichoderma Eg1 gene reaches 97% by comparison and analysis at NCBI, and the strain is also proved to have a key gene for synthesizing cellulase, namely endoglucanase gene Eg1, namely the capacity of producing cellulase.

Example 4: growth promoting effect on plants cultivated in common soil

The growth promotion effect of the HTTA-Z0002 strain on plants is tested by a conventional method, tomato seeds (a control) are respectively soaked in HTTA-Z0002 strain fermentation liquor and clear water which are diluted by 500 times, the tomato seeds are planted in a pot filled with sterilized soil, 3 tomato seeds are repeatedly treated in each pot, when 2 true leaves are treated, roots are irrigated for 1 time by the fermentation liquor with the same concentration, the cultivation is carried out for 25 days, and the tomato seeds are pulled out along with the roots.

TABLE 2 growth promoting action of HTTA-Z0002 strain on tomato seedlings

Tomato seedling	CK	Treatment of	The growth rate%
				Root length/cm	2.9	5.7	96.6
Plant height/cm	9.9	16.9	70.7
				Stem thickness/cm	0.7	1.3	85.7
Fresh weight/g	16.3	31.9	86.0
				Dry weight/g	2.1	4.8	128.6
Chlorophyll a/mg/g & FW	0.4	0.6	50.0
				Chlorophyll b/mg/g FW	0.3	0.6	100.0
Total chlorophyll/mg/g FW	0.4	0.9	125.0

As can be seen from Table 2, the HTTA-Z0002 strain has obvious growth promoting effect on the germination of tomato seeds and the growth of seedlings, and tomato plant seedlings treated by the bacterial liquid are stronger than tomato plant seedlings treated by clear water, have large leaves, dark green leaves, thick stems, long root hairs and 70% increase of the average plant height of the seedlings; the root length is increased by 96.6 percent; the stem thickness is increased by 85.7 percent, the chlorophyll a, the chlorophyll b and the total chlorophyll of the tomato leaves are respectively improved by 50.0 to 125.0 percent and 81.2 percent compared with the control, and the average fresh weight and the average dry weight are respectively increased by 86.0 to 128.6 percent.

Example 5: growth promoting effect on plants cultivated in saline soil and induction of plant salt resistance

Planting tomato seedlings in natural seaside saline soil (NaCl concentration is 0.4%), setting 2 controls of the tomato seedlings planted in common cultivated soil and Trichoderma-free saline soil, treating for 2, 4, 8 and 10 days, testing by a conventional method, comparing the length of radicle and hypocotyl with the controls, and calculating germination index; the seedling grows for 20 days, a complete plant is harvested, the stem thickness, the plant height, the root length, the number of the unfolded leaves and the like of the seedling are measured by a conventional method to calculate the seedling strengthening index, and simultaneously the contents of superoxide dismutase (SOD), Peroxidase (POD), Catalase (CAT), polyphenol oxidase (PPO), Phenylalanine Ammonia Lyase (PAL), proline, soluble sugar and the like related to stress resistance of the plant, and the contents of Na + and K + in the root and the leaf of the plant related to salt resistance are tested.

The result proves that the germination of the tomato seeds in the saline soil is inhibited, the radicle and the hypocotyl are short, the promotion effect of adding the HTTA-Z0002 strain on the germination of the tomato seeds is large, the radicle and the hypocotyl are close to normal, the germination index is obviously higher than that of a control, and the germination index is improved by 56.7%. The SOD, POD, CAT, PPO, PAL, proline and soluble sugar content of the plant are obviously increased by 100-180%

TABLE 3 influence of Trichoderma treatment on the germ root, hypocotyl and germination index of tomato seeds under salt stress

Lower case english letters indicate the significance of the difference between multiple comparisons α ═ 0.05, and between the same columns of data without the same letters.

As shown in table 4 below, the height, leaf length, expanded leaf number, and stem thickness of trichoderma harzianum treated plants were increased by 87.5%, 39.7%, 45.1%, and 69.0%, respectively, compared to the control, and the difference was significant compared to CK (P < 0.05).

TABLE 4 influence of Trichoderma treatment on tomato seedling morphology

TABLE 5 influence of Trichoderma treatment on Na + and K + content in tomato roots and leaves

As can be seen from Table 5, compared with the control, Na is present in the roots and leaves of the tomato after the HTTA-Z0002 strain treatment⁺The contents are reduced to different degrees, compared with the control, Na⁺The accumulation in roots and leaves is reduced by 43.1% -65.2%, and K in roots and leaves⁺Increased content of K in roots and leaves⁺The content is improved by 37.0 to 46.8 percent. After the tomato grows for 20 days, the water content of the control dry weight is 920.7%, the water content of the strain-treated dry weight is 1200.1%, the treatment can promote the absorption of tomato plants to water, keep the higher water content in the body, and reduce Na⁺Accumulation in plant body, increase K + content in plant body, and thus have different degreesThe salt damage is relieved or relieved, the growth condition of the tomato seedlings also shows that the leaf dehydration withered area of the control tomato seedlings is larger, and heavier salt damage symptoms are shown, and the salt damage symptoms of the second leaf of the tomato seedlings treated by the strain are obviously relieved and are reduced by 61.7 percent compared with the control salt damage index.

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core ideas. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims (2)

1. The application of trichoderma in promoting plant growth is characterized in that the trichoderma asperellum is trichoderma asperellum (trichoderma asperellum)Trichoderma asperellum ) Bacteria, strain number: HTTA-Z0002 with the preservation number of CGMCC No. 15677; the application comprises degrading phosphorus, potassium, calcium phytate or cellulose and promoting the germination of plant seeds and the growth of seedlings.

2. A fertilizer comprising the trichoderma as defined in claim 1.

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