CN1122445C - Plant resistance inductor - Google Patents

Abstract

A plant inducer, characterized in that the plant inducer mainly contains oligosaccharides, obtained by the following method: using Fusarium graminearum (F. graminearum Schw) as a microbial fermentation to obtain a large amount of mycelia body; the fermented mycelium is enzymatically degraded in a salt solution, the enzyme dosage is 0.5-3.0% by weight, the concentration of the salt solution is 0.01-0.5%, pH3.0-7.0, the reaction temperature is 15-60°C, and the reaction time is 1-10 Hours; heating, hot water extraction. The invention has high activity of inducing plant resistance and promoting plant growth and development, and has low production cost and little pollution, and is suitable for large-scale production.

Description

a plant inducer

The invention relates to microbial technology, and in particular provides a plant inducer with biologically active ingredients such as oligosaccharides obtained through microbial fermentation combined with enzymatic degradation under certain conditions, and is applied to crop disease prevention and control technologies such as soybeans and cotton.

Active oligosaccharides are a class of polymers composed of 2-15 monosaccharides with a certain structure and biological activity. It is generally believed that active oligosaccharides have the functions of regulating plant growth, development, reproduction, disease prevention and disease resistance, etc. , can stimulate the plant's immune system response, each active oligosaccharide can send out information to regulate specific functions, activate defense responses and regulate plant growth, produce active substances with anti-disease, and inhibit the formation of diseases, especially oligosaccharides from different sources Glycans can target different pathogenic bacteria (including ecological races), so that a series of oligosaccharide pesticides targeting various diseases can be developed to solve the problem of ecological variation of pathogenic bacteria that is difficult to solve by genetic engineering and genetic breeding. Most of the international research is the oligosaccharide fragments of plant and microbial cell wall polysaccharide degradation. More and more experimental results show that the plant cell wall not only acts as a structural barrier for defense, but also plays an active defense response when infected by pathogens. In the interaction between plants and pathogens, on the one hand, they secrete β-1,3-glucose, chitosanase and chitinase to directly inhibit the growth of fungi; on the other hand, they secrete endopolygalacturonase and Pectinases degrade the cell walls of plants. The availability of these oligosaccharins at the plant-pathogen interface with lyases confirmed that these fragments indeed function in vivo. These glycosidic bond cleavage enzymes can degrade the cell walls of plants and fungi into active oligosaccharins in vitro, so the regulatory mechanism of plants must involve inhibiting the partial activity of these enzymes so that they can produce oligosaccharins for a longer period of time. Conducive to the plant's disease resistance response. Therefore, inhibitors of glycosidic bond cleavage enzymes may play a role in the regulation mechanism of oligosaccharide induction. The enzymatic fragments released during this process - the cell wall oligosaccharides of pathogenic bacteria and plants, are likely to be the production mechanism of oligosaccharins in vivo. After the inducer is recognized by the host plant, the plant must produce second messengers to transmit the signal on the membrane receptor to the nucleus to result in the transcription of the gene. The difference between disease-resistant or susceptible plant varieties is not the difference in their defense system, but the difference in the quality or quantity of the initial recognition and subsequent signal molecule production. Oligosaccharides will be able to reduce this difference.

The preparation of existing plant inducers containing active oligosaccharides usually adopts the technology of plant extraction or plant tissue culture extraction. Due to the limitation of land area, four seasons, slow growth and other factors, large-scale batch production is almost impossible. impossible.

The object of the present invention is to provide a plant inducer, which has high activity of inducing plant resistance and promoting plant growth and development, and has low production cost and little pollution, and is suitable for large-scale production.

The invention provides a plant inducer, which is characterized in that the plant inducer mainly contains oligosaccharides, obtained by the following method:

(1) Using Fusarium graminearum (F. graminearum Schw) as the microbial fermentation to obtain a large amount of

of mycelium;

(2) ferment mycelium in _MgSO Salt solution with chitinase, cellulase, bromelain

One or more of enzymes, lysozyme, lipase, chitosanase for enzymatic degradation, the amount of enzyme

0.5-3.0% by weight, the concentration of the salt solution is 0.01-0.5%, pH3.0-7.0, reaction temperature

15-60℃, reaction time 1-10 hours;

(3) Heat up to 80-110°C, extract with hot water for 5-20 hours.

Fusarium graminearum Schw (F. graminearum Schw) described in the present invention is a kind of common wheat fungal pathogen, belongs to Ascomycetes, and the macroconidia of this fungus is born on the solitary nearly spherical lateral bottle-shaped stalk Or born on the bottle-shaped stalks with complicated branches, the stalks are 10-14um×3.5-4.5um. Large conidia are sickle-shaped, with obvious ventricle and back. Conidia are colorless, pink when aggregated. Light red to orange sticky substances (sticky conidia) are sometimes produced on the surface of the culture medium. The strains used in the present invention are common known strains, such as the strains adopted in the examples are respectively derived from the preserved bacterial strain F23 Fusarium graminearum of Jiangsu Academy of Agricultural Sciences Plant Protection Institute; China Culture Collection Center ACCC30213 Fusarium graminearum Strain (F. graminearum Schw); GF Fusarium graminearum from the Institute of Plant Protection, Shaanxi Academy of Agricultural Sciences. Bacterial strain used in the present invention can grow on general fungal culture medium, as: PDA medium (peeled and sliced potatoes 200g, boiled on low heat for 30 minutes, filtered, collected filtrate, added 2.0% glucose and water 1000ml) Cha's medium ( 2% sucrose, 0.1% KH ₂ PO ₄ , 0.05% KCl, 0.05% MgSO ₄ , 0.001% FeSO _{4 ,} 0.3% NaNO ₃ , PH6.7), but the biomass is different under different medium conditions.

In order to improve biomass, the preferred fermentation medium of the present invention is as follows:

 Peptone 0.3-2.0%,  Soybean flour 0.5-3.5%,

  Yeast powder 0.2-1.5%, Corn steep liquor 0.2-2.0%,

Sucrose 0.5-2.0%, KH ₂ PO ₄ 0.6-1.5%,

MgSO ₄ 0.1-1.0%, NaCl 0.01-0.5%.

When the plant inducer of the present invention is used to prevent and control plant diseases of soybean, tobacco, cotton and eggplant, the product can be soaked in seeds, sprayed on leaves or treated with root irrigation, and the use concentration is 10-2000ppm.

The present invention has the following advantages:

1. Using microorganisms with a certain cell wall structure, the active oligosaccharide raw material can be stably obtained through the fermentation process.

2. Oligosaccharide fragments with higher biological activity can be obtained by enzymatic hydrolysis.

3. The disease control applied to crops has the characteristics of high efficiency, non-toxicity, no pollution to the environment, and no resistance to pesticides.

4. To a certain extent, it can solve viral diseases, verticillium wilt and other crop diseases that have no effective pesticide control.

5. The source of raw materials is abundant, and the production cost is relatively low, which is conducive to the realization of industrialized production.

Below by example the technology of the present invention is further described:

Example 1 Fermentation of ACCC30213 Fusarium graminearum on different media

PDA: 20% peeled potatoes, (w/v) into strips, boiled on low heat for 30 minutes, filtered through eight layers of gauze, collected the filtrate, and added 2.0% glucose. F ₁ : peptone 0.3%, soybean powder 2.0%, yeast powder 0.2%, corn steep liquor 0.2%, sucrose 2.0%, corn starch 1.5%, KH ₂ PO ₄ 0.6%, MgSO ₄ 0.1%, NaCl 0.01%. F ₂ : peptone 0.3%, soybean powder 3.5%, yeast powder 0.2%, corn steep liquor 0.2%, sucrose 2.0%, KH ₂ PO ₄ 0.6%, MgS4 0.1%, NaCl 0.01%. After culturing for 10 days at 25°C and 80 rpm, the results of the cultivation were as follows: the biomass (dry weight of mycelia/fermentation liquid) cultured in medium PDA, F ₁ , and F ₂ were 0.57, 1.70, and 2.13, respectively.

Embodiment 2 Fermentation of GF Fusarium graminearum on different substratum

_F2 medium: peptone 0.3%, soybean powder 3.5%, yeast powder 0.2%, corn steep liquor 0.2%, sucrose 2.0%, KH ₂ PO ₄ 0.6%, MgSO ₄ 0.1%, NaCl 0.01%. _F3 medium peptone 0.3%, soybean powder 0.5%, yeast powder 0.2%, corn steep liquor 0.2%, sucrose 2.0%, corn starch 3.0%, KH ₂ PO ₄ 0.6%, MgSO ₄ 0.1%, NaCl 0.01%. _F4 medium: peptone 0.3%, soybean powder 0%, yeast powder 0.2%, corn steep liquor 0.2%, sucrose 3.5%, corn starch 1.0%, KH ₂ PO ₄ 0.6%, MgSO ₄ 0.1%, NaCl 0.01%. Cultivate for 10 days at 25°C, 80rpm. The fermentation results showed that the biomass of F ₂ medium was higher than that of medium F ₃ and F ₄ .

Example 3 F ₂₃ Fusarium graminearum fermentation scale-up culture test

In a 14L air-lift fermenter, inoculate 800mL of this strain into 12L of _F2 medium and culture it at a constant temperature of 25°C for 3 days. After 40 hours of cultivation, the biomass can reach 1.4%, especially in the first 24 hours. The fastest multiplication, slow proliferation after 40 hours, 3 days of cultivation compared with 40 hours, the biomass increased very little. This shows that the time required to obtain the same biomass is greatly shortened in the scale-up experiment than in the shaker culture. However, the maximum biomass that can be achieved by the scale-up experiment is smaller than that of the shaker culture. This may be due to differences in oxygen supply. Three batches of continuous cultivation were carried out in the scale-up test, and the results were basically repeated, and no bacteria were found, indicating that F ₂₃ Fusarium graminearum has strong competitive growth ability and grows rapidly, which is suitable for industrial production.

Example 4 Preparation of GF Fusarium graminearum and ACCC30213 Fusarium graminearum enzymatic hydrolyzate

Add 100mL of culture medium into a 250mL Erlenmeyer flask, inoculate the liquid strains of GF Fusarium graminearum and ACCC30213 Fusarium graminearum into the _F2 medium, the inoculum size is 10mL, and culture at 25°C and 80rpm shaker After 10 days, the mycelium was collected by centrifugation, rinsed with water, then vacuum filtered, dried, crushed and sieved to obtain bacterial powder. Gained bacterial powder is added in the magnesium sulfate of 0.6M respectively, add chitinase, chitosanase, cellulase compound enzyme enzyme by 1.0% (to substrate), react at 40 ℃, after the reaction finishes, in 100 Heat in a water bath at ℃ for 10 minutes to inactivate, extract at 70℃, centrifuge at 4000rpm to remove the precipitate, and obtain the enzymatic hydrolysis products of GF Fusarium graminearum and ACCC30213 Fusarium graminearum respectively.

Example 5 F ₂₃ Fusarium graminearum fermentation bacteria powder enzymatic degradation scale-up test

In a 25L stirred reactor, add 260 grams of fermented bacteria powder, 26 grams of compound enzymes of chitinase, chitosanase, cellulase, bromelain, and lipase, and an appropriate amount of 0.6M magnesium sulfate. 100rpm, temperature 37°C, enzymatic hydrolysis for 3 hours. It was found that the enzymolysis solution obtained from the scale-up test induced wheat PAL (phenylalanine ammonia lyase) activity (characterizing plant disease resistance index) to be consistent with that in the 100mL reaction bottle (Table 1), which indicated that F23 Fusarium graminearum powder enzymolysis The amplification effect of the reaction is small.

     Table 1 Enzyme Liberation Test and the PAL Activity of the Shake Flask Reaction

 Enzymatic hydrolysis reaction PAL activity increase/control (%)

                                                                    

                                                                               

Example 6 Effect of F ₂₃ Fusarium graminearum Enzyme Degradant on Plant PAL Resistance Enzyme Biological Activity

After cleaning the wheat and soybean seeds, add 8% NaC10 to disinfect them for 10 minutes, rinse them with sterile water for 3 times and set aside. Cotton seeds were delinted with concentrated sulfuric acid, washed with water and sterilized with 8% NaC10 for 30 minutes, rinsed with sterile water three times, soaked in warm water at 55°C for 30 minutes, and dried for later use.

Take 0.4ml of the enzyme extract in a clean test tube, add 2ml of cold 0.1M boric acid buffer solution (PH8.8, containing 5mmol/L mercaptoethanol). Add 1ml of 20mM L-phenylalanine to the extract, add 1ml of water to the control, mix well, react in a water bath at 35°C for 45 minutes, and then measure the inactivation, using the reaction product - trans-cinnamic acid (t-cinnamic acid) The OD ₂₉₀ per hour is 0.01 as one activity unit (U).

Compared with the control, the activity of phenylalanine ammonia lyase (PAL) in the seedlings of wheat, soybean, and cotton seeds treated with different concentrations increased in different degrees. F ₂₃ F 23 Fusarium graminearum enzyme degradation product six concentrations of wheat PAL activity were higher than the control, of which 20ug/ml and 40ug/ml concentration of the best effect. Three concentrations of F ₂₃ Fusarium graminearum enzyme degradation products were used to treat soybean PAL, and 50ug/ml was the best, so the activity of PAL did not increase much compared with the control, but the range of activity concentration should be between 10-100ug/ml.

Example 7 Application experiment of GF Fusarium graminearum enzymatic hydrolyzate in the control of cotton verticillium wilt

For each treatment with 800g of seeds, add 1600ml of medicinal solution to soak the cotton seeds for 24 hours, and soak the seeds in water for 24 hours as a control, and stir twice during the soaking to ensure uniform treatment. After the treatment, remove the seeds and drain the water, sow them in nutrient pots, 3 seeds per pot, 1000 nutrient pots per treatment, set up plots according to 5 treatments, and investigate the emergence of each treatment. The nutrient pot seedlings were moved to the cotton verticillium wilt recurrent field, each treatment area was 50 square meters, repeated 4 times, and randomly arranged. In mid-April, the emergence and control of seedling diseases were investigated, and in the first ten days of September, the incidence of Verticillium wilt was investigated.

The test results showed that GF Fusarium graminearum powder enzyme extract had a certain control effect on cotton verticillium wilt, and the control effect was different with different application methods and concentrations. The control effect of 50ug/ml treatment at the seedling stage was the highest at 63.99%, and the control effect of 800-fold drug solution of mobuzin-methyl was 43.98%. Sectional surveys were 65.82% and 50%. Seed soaking method, seedling investigation, 50ug/ml treatment has the highest control effect of 100%, and the control effect of 800 times of methyl mobzin is 55.5%. The pole section surveys were 81.8% and 63.65% respectively. The effect of soaking seeds is better than that of spraying.

Example 8 The control effect of Fusarium graminearum (ACCC30213) enzymatic hydrolyzate preparation on soybean mosaic

The present embodiment adopts Fusarium graminearum (ACCC30213) as fermentation strain, presses the fermentation condition of F ₂₃ Fusarium graminearum, the enzymatic hydrolyzate preparation of the mycelium that obtains has carried out soybean mosaic disease in plant protection station of Heilongjiang Province In the standardized field experiment, the control effect reached 70%, and the yield increased by 13%.

Example 9 Effect of F ₂₃ Fusarium graminearum Enzyme Degradant on Plant Endophytes

Sow the cotton seeds delinted by sulfuric acid in a nutrient bowl with a diameter of 15 cm, and spray with F ₂₃ Fusarium graminearum enzyme degradation product (diluted 200 times) after the cotton emerges, spraying once every 3 days. Spray 5 times in a row. After the last spraying, the cotton stems were separated at different times. Investigate after a large number of dead seedlings appear in the control group. Take spraying water as a control. The beef peptone medium was selected, and the gradient dilution plate method was used for separation. The experimental results are as follows:

Effect of F ₂₃ Fusarium graminearum Enzyme Degradation Products on Bacterial Community in Cotton Stem deal with Dilution factor 10 days after spraying (cfu/g) 30 days after spraying (cfu/g) 45 days after spraying (cfu/g) Degradants 200 8.0×10 ⁷ 5.0×10 ⁷ 1.8×10 ¹⁰ CK 4.2×10 ⁹ 3.0×10 ⁸ 2.0×10 ¹⁰

It can be seen from the table that the number of bacterial communities in cotton stems decreased significantly after spraying oligosaccharides, and then rose again in the later period.

Claims (3)

1, a kind of plant induced resistance agent is characterized in that this plant induced resistance agent mainly contains active oligomeric sugar, obtains by following method: (1) is that the bacterial classification microbial fermentation obtains a large amount of mycelium with F.graminearum schw F.graminearum Schw; (2) with fermentation mycelium at MgSO ₄Degrade with one or more compound enzymes that carry out of chitinase, cellulase, bromelain, lysozyme, lipase, chitosan enzyme in the salting liquid, every kind of enzyme proportion is 1%--51%, enzyme dosage 0.5-3.0% weight, the concentration of salting liquid is 0.01-0.5%, pH3.0-7.0, reaction temperature 15-60 ℃, reaction time 1-10 hour; (3) be warming up to 80-110 ℃, hot-water extraction 5-20 hour.

2, according to right 1 described plant induced resistance agent, it is characterized in that: described inoculation is in the fermentation medium of following prescription:

Peptone 0.3-2.0%, analysis for soybean powder 0.5-3.5%,

Dusty yeast 0.2-1.5%, corn steep liquor 0.2-2.0%,

Sucrose 0.5-2.0%, KH ₂PO ₄0.6-1.5%,

MgSO ₄ 0.1-1.0％， NaCl 0.01-0.5％。

3, the described plant induced resistance agent of claim 1 is used to prevent and treat soybean, tobacco, cotton, eggplant plant disease, it is characterized in that with this product seed soaking, foliage-spray or root irrigation, working concentration is 10-2000ppm.