LU503323B1

LU503323B1 - Preparation method and application of Trichoderma-containing inducer

Info

Publication number: LU503323B1
Application number: LU503323A
Authority: LU
Inventors: Xinhua Wang
Original assignee: Univ Shanghai Jiaotong
Priority date: 2023-01-09
Filing date: 2023-01-09
Publication date: 2023-07-10

Abstract

This invention provides a preparation method and application of Trichoderma-containing inducer, comprising Trichoderma, a carrier and a cross-linking agent, and the inducer is prepared by embedding according to a certain proportion. The stress tolerance of corn sprouts was improved by inducing corn seeds. The Trichoderma inducer can effectively protect Trichoderma from being damaged by harsh natural environment such as exposure to strong sunlight and flooding, and improve the survival rate and biological activity of biocontrol bacteria in soil. Moreover, the Trichoderma inducer can induce corn and other crop seeds to produce a variety of substances related to plant stress tolerance after germination, which can improve the growth capacity, yield and quality of corn. The method is simple to apply, the products prepared by this method are environment-friendly and pollution-free, making this invention have high application and popularization value.

Description

DESCRIPTION LU503323

Preparation method and application of Trichoderma-containing inducer

TECHNICAL FIELD

[01] This invention belongs to the field of plant protection, particularly relates to preparation method and application of Trichoderma-containing inducer.

BACKGROUND

[02] Corn is an important cereal crop in the world. It is also an important grain, feed and industrial raw material crop in China, and its total output ranks second only after rice. At present, the main diseases that seriously affect corn production are corn northern leaf blight (Dreschera turcica), corn southern leaf blight (Bipolaris maydis), Corn smut (ustilago maydis), corn stalk rot (Fusarium graminearum), etc. Traditional pesticides and fertilizers have increasingly negative effects on agricultural systems, therefore, partial or complete use of biological control agents or biological methods has a positive effect on solving the above problems.

[03] In the process of long-term interaction between plants and various pathogens in nature, plants have formed many levels of defense mechanisms to resist the harm of external adverse environment to them.

[04] Plant defense system can be divided into three levels: constitutive defense, innate immunity and systemic immunity. Constitutive defense was formed in the early stage of plant evolution, which constituted the first line of defense for plants to resist the infection of pathogens, and could block the infection of most pathogens. Plant defense system can be divided into three levels: constitutive defense, innate immunity and systemic immunity. Constitutive defense was formed in the early stage of plant evolution, which constituted the first line of defense for plants to resist the infection of pathogens, and could block the infection of most pathogens. Therefore, tolerance inducer can make corn seedlings produce stress-tolerance substances, hence improving their tolerance to soil-borne diseases, salt stress, drought and flood, etc., the method of using inducer is beneficial to seedling survival and plant growth, and can reduce the use of chemical pesticides and fertilizers, making no pollution to the atmosphere and soil and no harm to natural enemies of insects.

[05] Treating corn seeds can induce seedlings to have defensive reaction, enhance the ability of seedlings to resist pathogen infection, accelerate the growth and development of Cron 503323 increase crop yield and improve crop quality. China has a long history of using seed treatment agents. In ancient times, seeds were soaked in warm liquid and soaked in medicine, which was recorded in the agricultural book "Book of Agricultural Victory" in the Western Han Dynasty.

The function of seed coating agent is mainly through active ingredients, while the function of inactive ingredients is to promote the active ingredients to better realize their functions. The main research direction of biological seed coating agent is to use active ingredients of living microorganisms or microbial metabolites. However, there are some problems exist in modern microbial seed coating agents: China has a vast territory, and the climatic conditions, soil conditions and environment differ from each other in different regions. The application effect of biological seed coating agents is easily affected by the field environment or can't be shown in a short time. For example, the biological seed coating agents that use Pseudomonas fluorescens as the main component have high requirements for temperature and humidity, and the fungal activity performance is different under varied temperature and humidity conditions.

[06] Microbial immobilization technology can fix cells in a limited space area by physical or chemical means, while keeps their original microbial activity. Microbial immobilization technology not only has the advantages of good stability, simple operation and easy realization, but can avoid the influence of unfavorable factors in the external environment on cells to a certain extent, which is conducive to the growth and reproduction of cells. According to the types of carriers and the combination of carriers and microorganisms, microbial immobilization methods can be divided into adsorption, cross-linking, embedding and composite immobilization.

Among the these methods, the embedding method is to embed microorganisms in the gel grid of high polymer or the semi-permeable membrane of high polymer, so that small molecular substrates and products can freely diffuse through the high polymer or semi-permeable membrane, which has little influence on microorganisms.

SUMMARY

[07] The purpose of this invention is to provide a preparation method and application of

Trichoderma-containing inducer, selecting appropriate biocontrol 7richoderma and microbial fixed embedding technology. The obtained Trichoderma-containing inducer has stable effect,

and is not affected by soil environment, temperature and humidity, pH, etc., and can induce CO 503323 and other crop seedlings to produce stress-tolerance substances, thus improving crops' tolerance to soil-borne diseases, salt stress, drought and flood, etc.

[08] In order to achieve the purpose described above, this invention provides the following solutions:

[09] This invention provides a Trichoderma-containing inducer comprising 7richoderma, a carrier and a cross-linking agent; the 7richoderma is Trichoderma atroviride CCTCC No. M 20221135, and the carrier 1s sodium alginate, chitosan and carrageenan. The limitation caused by using only one carrier can be overcome by adopting the technical solution. Sodium alginate, as a carrier, has the advantages of non-toxicity to microorganisms, good mass transfer, low price, easy operation and widely used, however, it also has the disadvantages of low mechanical strength and easy decomposition by microorganisms in soil. The molecular structure of chitosan and carrageenan determines their stable performance, which can improve the strength of embedded particles, make them easy to fix, and have high microbial survival rate and reproduction rate. Moreover, chitosan can also provide carbon source for microbial reproduction and growth.

[010] Further, the crosslinking agent is calcium chloride solution.

[011] On the other aspect, this invention provides a preparation method of

Trichoderma-containing inducer, comprising the following steps: step1, inoculating 7richoderma atroviride strain CCTCC No. M20221135 into PDA culture medium, culturing in light incubator at 25-30°C for 3-5d, preparing spore suspension, inoculating Trichoderma spores with a concentration of 1x10°-3x10° /ml Trichoderma spores in PD, and at 25-30°C and centrifuging at 200rpm, culturing for 4-5 days to obtain fungal solution, next, centrifuging at 8000-10000rpm for 20 mins to obtain mycothallus; step 2, taking sodium alginate, chitosan and carrageenan as carriers, sterilizing, and embedding the mycothallus into the carrier to obtain a thallus-carrier mixture; step 3, dropping the thallus-carrier mixture into a sterile cross-linking agent by physical extrusion, making the mixture to balls, washing it with sterile physiological saline for 8-10 times to obtain Trichoderma atroviride CCTCC No. M 20221135 embedded beads, drying at 27°C for 18-24h, and storing at 4°C.

[012] Further, the carrier is a carrier solution with a mass concentration of 2-2.5% formed, 503323 by dissolving sodium alginate, chitosan and carrageenan in sterile water; The dosage ratio of carrier solution to mycothallus is 10ml:1g; the mass ratio of sodium alginate, chitosan and carrageenan in the carrier is 1:3:2.

[013] Further, the number of effective viable bacteria in the fungal solution is more than 1x10° /ml.

[014] Further, the carrier is sterilized under the conditions of 121°C and 0.12MPa for 20min.

[015] Further, the crosslinking agent is calcium chloride solution with mass concentration of 1%; the dosage ratio of calcium chloride solution to mycothallus is 20ml:1g.

[016] Further, the solidification time of the embedded beads is 18h-24h.

[017] On the third aspect, the invention relates to the application of Trichoderma-containing inducer in inducing corn and other crop sprouts to produce stress-tolerance substances. Corn seeds were induced and colonized by Trichoderma-containing inducer.

[018] According to the invention, the Trichoderma atroviride is preserved in China Center for Typical Culture Collection (CCTCC) on July 19, 2022, and the preservation address is

No.299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC No. M 20221135.

[019] Compared with prior art, this invention has the following beneficial effects:

[020] 1. The invention adopts Trichoderma-containing inducer to induce corn seed sprouts, so that corn sprouts can produce stress-tolerance substances, and improve crop stress tolerance to soil-borne diseases, salt stress, drought and flood, etc., and improve its yield and quality. The method is safe and nontoxic, it reduces the use of chemical pesticides and chemical fertilizers, thus meeting the development requirements of green agriculture and carbon emission reduction.

[021] By embedding the Trichoderma-containing inducer, the invention avoids the defects that a large number of mycothallus could die, if they are directly exposed to the open and changeable soil environment after being applied to the soil, and that it is difficult to form dominant flora and exert the due effect of the microbial inoculum.

[022] The invention screens and combines the embedding carriers, which overcomes the disadvantages of weak mechanical strength, low microbial survival rate and the like caused BY)503323 single use of one carrier. After being applied to the soil, it has impact resistance in the natural environment, etc., and plays a better role in protecting microbial active ingredients.

[023] The Trichoderma-containing inducer of the invention can induce corn sprouts to produce a variety of stress-tolerance substances such as malondialdehyde, calcium ion, jasmonic acid and salicylic acid, in order to improve the adaptability of crops to drought, saline-alkali and other adverse environments. The product is simple in preparation method, convenient to use, safe and reliable, and has good application potential and value.

DETAILED DESCRIPTION OF EMBODIMENTS

[024] The invention will be described in detail with reference to the following examples.

The following examples will help those skilled in the art to further understand the present invention, but will not limit the present invention in any way. It should be pointed out that for those skilled in the art, several adjustments and improvements can be made without departing from the concept of the present invention. These all belong to the scope of protection of the present invention.

[025] (1) Preparation of mycothallus

[026] Trichoderma atroviride strain CCTCC No. M 20221135(D1) was inoculated into

PDA culture medium, cultured in 28°C light incubator for 4 days, and spore suspension was prepared. Trichoderma spores with a concentration of 1x10° /ml were inoculated into PD, and cultured at 28°Cand 200rpm for 5 d to obtain a fungal solution with an effective viable count of 1x10” /ml. After centrifugation at 10000rpm for 20 min, the fungal cells were obtained.

[027] (2) Preparing a thallus-carrier mixture

[028] Preparation of mixed carrier of sodium alginate, chitosan and carrageenan. Sodium alginate, chitosan and carrageenan were used to prepare 10ml carrier solutions Z1, Z2, Z3 and

ZA according to different mass ratios, and the carrier mass concentration was 2%. (See Table 1 for details). After steam sterilization at 121°C and 0.12MPa pressure for 20min, lg of the above-mentioned bacteria were added respectively, and they were shaken and evenly mixed on a constant-temperature magnetic stirrer.

[029] Table 1 Composition of carriers in different proportions oe | 8 | 8 VE) os UN JS y EE) os rr fr 0 8

[030] (3) Cross-linking embedding fixation

[031] The above-mentioned fungal-carrier mixture was evenly dropped into 20mL of cross-linking solution containing 1% CaCl, with a pipette at a certain speed, with the volume of each drop being 0.02ml, cross-linked for 18h, the particles were filtered out, and washed with sterile physiological saline for 10 times, then obtained respectively the embedded particles of

Trichoderma atroviride CCTCC No. M 20221135, namely inducers Y1, Y2, Y3 and Y4, and drying them at 27°Cfor 24h, and preserved at 4°C.

[032] 2. Performance test

[033] (1) Measurement of mechanical strength of inducer particles

[034] Four inducer particles Y1, Y2, Y3 and Y4 were taken with the same size, and they were put on a glass plate in a square shape, a glass dish was put on it, and slowly added the weights into the glass dish until the particles were completely deformed. Recorded the glass dish that made the particles completely deformed, also recorded 1/4 of the total weight of the added weights is the bearing weight of each embedded particle, which indicates the mechanical strength of the pellets. Each treatment was repeated for 3 times, and the results were averaged.

[035] Table 2 Determination of mechanical strength of different proportions of carrier inducer particles

HMEIERERES

[036] (2) Determination of viable bacteria number of inducer particles

[037] 1g of fresh embedded particles were weighed, and were put into 0.2 mol /L sodium citrate solution until they reached a constant volume of 10 mL, then, fully oscillated with a vortex oscillator until the particles were completely dissolved, so that all the bacteria were dissolved out, then cultured at 28°C for 3d with dilution plate method, and counted the colonies 503323

Each treatment was repeated for 3 times, and the results were averaged. Each index is calculated according to the following formula.

[038] The number of viable bacteria (cfu /g) = (the number of viable bacteria at a certain dilution x dilution times x fungal liquid volume)/particle weight

[039] Table 3 Determination of viable bacteria number of vector inducer particles in different proportions

Granular bacteria wean PO LOTO

[040] From tables 2 and 3, it can be seen that the mechanical strength of inducer Y1 was the highest, reached 50.9g/g, and the number of granular viable bacteria was the highest, reaching 13x10®cfu/g, indicating that the inducer of carrier Z1 with the mass ratio of sodium alginate, chitosan and carrageenan of 1: 3: 2 had the best performance.

[041] 3. Induced stress resistance test

[042] The surface of corn seeds was disinfected with 75% ethanol for 10min, and then dried in a super clean table. According to the mass ratio of Trichoderma inducer Y1 and seeds of 10: 1, they were weighed into a beaker, and shaken until they were well mixed, and colonized them.

The control was uninduced corn seeds.

[043] (1) Investigation of germination rate

[044] The tested samples were placed in a dark incubator at 28°C, and the germination number was counted every day.

[045] Germination rate% = germination number/total number of cornx 100%

[046] (2) Effect on the growth of maize seedlings

[047] The induced seeds were sowed in flowerpots, 10 seeds in each pot, three repetitions in each treatment, and the uninduced seeds were taken as the control. The germination rate was calculated after 7 days, and the root length, root number, plant height, fresh weight and d1Y)503323 weight of plants were measured after 14 days. See Table 4.

[048] Table 4 Determination of different indexes germination .

Length of | Number of | Height of | Plant fresh | Plant dry

Process percentage root (cm) root stalk (cm) | weight (g) | weight (g) (%)

Induction 86.6 20.6 12 29.4 1.7 0.27 treatment

Le me LL

[049] It can be seen from Table 4 that the germination rate, root length, root number, plant height, fresh weight and dry weight of corn seeds treated with inducer are higher than those without inducer containing Zrichoderma. The results showed that Trichoderma-containing inducer was beneficial to the germination of corn seeds and the absorption of nutrients, and it grew well.

[050] (3) Determination of stress resistance index of maize sprouts.

[051] Malondialdehyde, calcium ion, jasmonic acid, salicylic acid and other indexes related to plant stress resistance were measured in seed sprouts after 7 days of treatment.

[052] Table 5 Determination of seed stress tolerance index of different treatments salicylic

Treatment MDA (nmol/l) calclum ion% jasmoniepmol/l acidpmol/l

[053] Malondialdehyde (MDA) is an organic substance produced by peroxidation of lipid in, 503323 tissues or organs of plants due to aging or damage in adversity. The starch in plants can be converted into glucose, because the freezing point of sugar solution is relatively low, so the body fluid of plants is not easy to coagulate in a low-temperature environment, hence adapting the cold environment. The stress resistance of plants can be judged by measuring the content of malondialdehyde in plants.

[054] Calcium is an essential element for plant growth and development, and it also acts as an important messenger to regulate the plant's resistance to environmental stress. The increase of calcium ion content indicates the enhancement of plant regulation ability under environmental stress.

[055] Jasmonic acid and its derivatives are lipid-based plant hormones, which can regulate various plant processes from growth and photosynthesis to reproductive development.

Particularly, jasmonic acid is essential for plants toprotect themselves from herbivores and harsh environmental conditions, as well as other types of abiotic and biological challenges.

[056] Salicylic acid is one of the endogenous signal molecules ubiquitous in plants. It is recognized as a plant hormone with important physiological functions. Studies have shown that salicylic acid has obvious effects on plant disease resistance, low temperature resistance, drought resistance and salt resistance, as well as fruit ripening, product preservation and seed germination.

[057] It can be seen from Table 5 that the contents of malondialdehyde, calcium ion, jasmonic acid and salicylic acid in corn seed sprouts induced by Trichoderma were higher than those in the control. The results showed that the corn sprouts treated with Trichoderma inducer contained higher plant stress-tolerance substances, which could better adapt to the growth in adverse environment, thus improving the yield and quality of corn.

[058] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which will not affect the essential content of the present invention.

Claims

CLAIMS LU503323

1. A Trichoderma-containing inducer comprising 7richoderma, a carrier and a cross-linking agent; the Trichoderma is Trichoderma atroviride CCTCC No. M 20221135, and the carrier is sodium alginate, chitosan and carrageenan.

2. A Trichoderma-containing inducer as claimed in claim 1, wherein, the crosslinking agent is calcium chloride solution.

3. A preparation method of Trichoderma-containing inducer, comprising the following steps: stepl, inoculating Trichoderma atroviride strain CCTCC No. M20221135 into PDA culture medium, culturing in light incubator at 25-30°C for 3-5d, preparing spore suspension, inoculating Trichoderma spores with a concentration of 1x10°-3x10° /ml Trichoderma spores in PD, and at 25-30°C and centrifuging at 200rpm, culturing for 4-5 days to obtain a fungal solution, next, centrifuging at 8000-10000rpm for 20 mins to obtain mycothallus; step 2, taking sodium alginate, chitosan and carrageenan as carriers, sterilizing, and embedding the mycothallus into the carrier to obtain a thallus-carrier mixture; step 3, dropping the thallus-carrier mixture into a sterile cross-linking agent by physical extrusion, making the mixture to balls, washing it with sterile physiological saline for 8-10 times to obtain Trichoderma atroviride CCTCC No. M 20221135 embedded beads, drying at 27°C for 18-24h, and storing at 4°C.

4. The preparation method of Trichoderma-containing inducer as claimed in claim 3, the carrier is a carrier solution with a mass concentration of 2-2.5% formed by dissolving sodium alginate, chitosan and carrageenan in sterile water, The dosage ratio of carrier solution to mycothallus is 10ml:1g; the mass ratio of sodium alginate, chitosan and carrageenan in the carrier is 1:3:2.

5. The preparation method of Trichoderma-containing inducer as claimed in claim 3, the number of effective viable bacteria in the fungal solution is more than 1x10° /ml.

6. The preparation method of Trichoderma-containing inducer as claimed in claim 3, in step 2, the carrier is sterilized under the conditions of 121°C and 0.12MPa for 20min.

7. The preparation method of Trichoderma-containing inducer as claimed in claim 3, the crosslinking agent is calcium chloride solution with mass concentration of 1%; the dosage ratio of calcium chloride solution to mycothallus is 20ml:1g.

8. The preparation method of Trichoderma-containing inducer as claimed in claim 3, the 503323 physical extrusion method is to uniformly drop the microbial agent-carrier mixture into the cross-linking agent with a pipette, and the volume of each drop is 0.02ml.

9. The preparation method of Trichoderma-containing inducer as claimed in claim 3, the solidification time of the embedded beads is 18h-24h.

10. Trichoderma-containing inducer as claimed in claim 1 or claim 3, the Trichoderma-containing inducer prepared by the method can be used for inducing corn and other crop sprouts to produce stress-tolerance substances.