CN114403146B - Application of pullulan in preparation of plant resistance inducer, plant resistance inducer and method - Google Patents

Abstract

The invention discloses an application of pullulan in preparing plant resistance inducer, plant resistance inducer and method, wherein the application range of the plant resistance inducer comprises induction of plant disease resistance, induction of plant stress resistance, antivirus and antifungal. The invention utilizes biological activity determination, physiological and biochemical test and other methods to determine the plant disease resistance induction activity and plant stress resistance induction activity of the pullulan, prepares plant resistance inducer, biological hormone, plant virus resistance agent and bactericide which take the pullulan as active ingredients, and is applied in fields.

Description

Application of pullulan in preparation of plant resistance inducer, plant resistance inducer and method

Technical Field

The invention belongs to the field of agriculture, and particularly relates to an application of pullulan in preparing a plant resistance inducer, a plant resistance inducer and a method.

Background

Biffen found that there was a disease resistance gene in wheat for the first time in 1905. In 1955, flor proposed a "gene-to-gene" hypothesis in the study of the specificity of the rust race, illustrating the unaffinity relationship between the disease-resistant genes of plants and the non-toxic genes of pathogens, on the basis of which the concept of "plant immunity" was developed and perfected. At present, the green means for fully utilizing the autoimmune defense function of plants to perform plant protection plays a positive role in disease-resistant breeding and comprehensive disease prevention and control, and has wide prospect. It is the plant immune elicitor (plant immune activator) that is a substance that does not have direct bactericidal or antiviral activity or is very low in activity, but that stimulates the plant's immune defenses by itself or its metabolites, thereby promoting the plant itself to produce disease resistance to the pathogen's system. Compared with the traditional pesticide, the plant immunity inducing and resisting agent has the characteristics of broad spectrum, low toxicity, no drug resistance risk, no lag and long lasting period, synergistic effect or synergy with other bactericides, and the like, promotes the plant protection to return to the utilization of the essential characteristics of the plant, and is a green ecological pesticide in the true sense.

Because the plant immunity inducer has no pesticide activity, but can induce plant disease resistance and insect resistance, the plant immunity inducer can be registered in various pesticide categories in pesticide management in China. Such as amino oligosaccharin, lentinan, etc. are registered as germicides, plant resistance inducers in sequence; s-elicitors were registered as plant growth regulators; meanwhile, the plant immunity inducer can be compounded with various pesticides, such as amino oligosaccharin and fosthiazate, and is registered as nematicide, tebuconazole, moroxydine hydrochloride, kasugamycin, trifloxystrobin and the like, and is registered as bactericide, and is compounded with 24-epibrassinolide and 28-Gao Yuntai lactone, and is registered as plant growth regulator.

The plant immunity inducer can also be used as biological hormone. The biological hormone is a series of organic compounds, inorganic compounds or microorganism products which can improve the nutrition and health condition of crops, improve the utilization rate of pesticides and fertilizers, stimulate the natural physiological process of crops, act on plants, seeds or soil, improve the stress resistance of the crops and finally improve the yield and quality of the crops. Biostimulant products can be classified as microbial agents and extracts thereof, protein hydrolysates, humic acid, seaweed and plant extracts, inorganic and synthetic products. In the market of China, the biological hormone products with larger sales amount are mainly humic acid products, amino acid products, alginic acid products and microbial fermentation products.

Pullulan is an extracellular polysaccharide secreted by fungi of the genus Aureobasidium, the most studied of which is Aureobasidium pullulans (a. Pullulans). Aureobasidium pullulans is a polymorphic fungus and has a plurality of forms such as polynuclear mycelium, chlamydospores, small elliptic yeast cells and the like. Aureobasidium pullulans can produce various metabolites, mainly including extracellular polysaccharide, single cell protein, enzyme, antibiotics and the like. Wherein, pullulan is the main extracellular polysaccharide produced by fermentation of Aureobasidium pullulans. The polysaccharide is a linear polysaccharide formed by polymerizing alpha-1, 6 glycosidic bonds of maltotriose repeated units connected by alpha-1, 4 glycosidic bonds, and has a molecular weight of 2-200 ten thousand, and is also called pullulan, aureobasidium polysaccharide, pullulan and pullulan. The polysaccharide has two important properties: structurally, the material is rich in elasticity and relatively high in solubility. The pullulan has strong film forming property, gas barrier property, plasticity and viscosity, has excellent characteristics of easy water dissolution, no toxicity, no harm, no color, no smell and the like, and has been widely applied to the fields of medicine, food, light industry, chemical industry, petroleum and the like. On day 19, 5 and 2006, the national institutes of health issued bulletin No. 8, pullulan is one of four new food additive products that can be used as a film coating and thickener in confections, chocolate coatings, diaphragms, complex flavourings and juice drinks.

Disclosure of Invention

The invention utilizes biological activity determination, physiological and biochemical test and other methods to determine the plant disease resistance induction activity and plant stress resistance induction activity of the pullulan, prepares biological source pesticide and biological stimulation hormone which take the pullulan as active ingredients, and is applied in fields.

The invention discloses a method for preparing a plant attractant by using pullulan and application of the pullulan in agricultural production. The application range comprises induction of plant disease resistance activity and induction of plant stress resistance activity. The plant resistance inducer provided by the invention can induce and improve the resistance of plants to fungal diseases, bacterial diseases, nematode diseases, pests and virus diseases and high-temperature, low-temperature and drought stress conditions.

The invention discloses an application of pullulan polysaccharide in preparing plant resistance inducer, wherein the application range of the plant resistance inducer comprises induction of plant disease resistance or induction of plant stress resistance.

The method specifically comprises the following steps:

the application of pullulan in preparing plant resistance inducer includes inducing plant to resist disease or inducing plant to resist reverse.

Optionally, the pullulan is linear polysaccharide formed by polymerizing a maltotriose repeating unit connected with alpha-1, 4 glycosidic bonds through alpha-1, 6 glycosidic bonds, and the molecular weight is 2-200 ten thousand daltons; the plant disease resistance comprises plant virus disease, plant bacterial disease and plant mycosis; the plant stress resistance comprises cold resistance and drought resistance.

The plant resistance inducer comprises pullulan and an auxiliary agent, wherein the preparation is in the form of soluble powder, water dispersible granule, soluble liquid, aqueous emulsion and microemulsion and all agriculturally acceptable preparation types; the content of the pullulan is 1-100% by mass percent.

The plant resistance inducer comprises pullulan and an auxiliary agent, wherein the preparation is in the form of soluble powder, water dispersible granule, soluble liquid, aqueous emulsion and microemulsion and all agriculturally acceptable preparation types; the content of the pullulan is 0.1-80% by mass percent.

The plant resistance inducer comprises pullulan and an auxiliary agent, wherein the preparation is in the form of soluble powder, water dispersible granule, soluble liquid, aqueous emulsion and microemulsion and all agriculturally acceptable preparation types; the content of pullulan is 30% by mass.

A pesticide composition comprising pullulan and a mixture of another or more commercial pesticides, wherein the content of pullulan is 0.1-99% by mass; the commercial pesticides include plant elicitors, fungicides, bactericides, antiviral agents, insecticides, nematicides, acaricides and combinations thereof.

A biostimulant comprising pullulan; the content of the pullulan is 0.1-100% by mass percent.

An anti-plant virus agent comprising pullulan; the content of the pullulan is 0.1-100% by mass percent.

A biocide comprising pullulan; the content of the pullulan is 0.1-100% by mass percent.

A method of enhancing the immunity of a plant and protecting the plant from pests, the method comprising the steps of: the product of the invention is applied to at least one of the area adjacent to the plant, the soil suitable for supporting plant growth, the root of the plant and the foliage.

The induction of plant disease resistance activity mentioned in the invention is essentially to improve the disease resistance capability of plants themselves, is a broad-spectrum disease resistance activity, and can be used for enhancing the disease resistance capability of plants to bacterial, fungal, viral, oomycete and nematode infection. The stress-resistance activity of the induced plants is essentially stress-resistance potential of the induced plants, and can be used for enhancing drought resistance, cold resistance, salt resistance, disease resistance and the like of the plants under the stress of biological factors such as insect pests, weeds and the like, temperature, moisture, salt and alkali, chemical factors, weather and other physical and chemical factors. The plant resistance inducer is a pesticide, can be called as a plant immunity activator and the like, has no direct bactericidal activity, can control and prevent the invasion of fungi, bacteria, viruses, nematodes, insects and other harmful organisms on plants by inducing the plant to generate disease resistance activity or stress resistance activity, has the advantages of difficult generation of resistance of pathogenic bacteria, relatively wide control spectrum, capability of being mixed with chemical agents and the like, and is a pesticide meeting the requirements of green prevention and control. The biological stimulus mentioned in the present invention is neither a pesticide nor a traditional fertilizer. The target of the biological hormone is the crop itself, which can improve the physiological and biochemical states of plants, the pesticide effect and the fertilizer utilization rate, the stress resistance level of the crop, and the final yield and the agricultural product quality of the crop.

Drawings

The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate the disclosure and together with the description serve to explain, but do not limit the disclosure. In the drawings:

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of pullulan;

FIG. 2 is a nuclear magnetic resonance carbon spectrum of pullulan;

FIG. 3 shows the variation of the expression level of the disease-resistant related gene after spraying Shi Pulu orchid polysaccharide on tobacco leaves.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The invention discovers for the first time that the pullulan has remarkable plant disease resistance induction activity and can be developed into novel biological source pesticides and biological stimulation hormones. As a natural polysaccharide with no toxicity, no harm and wide resources, the invention has important value and significance for the development and utilization of the activity of pullulan pesticides and the green prevention and control of plant diseases and insect pests.

The compound pullulan of the present disclosure may be obtained by fermentation, and preferably, the microorganism that can be fermented to produce pullulan is Aureobasidium pullulans. The biological activity measurement result and the physiological and biochemical experiment result in the embodiment of the invention prove that the pullulan polysaccharide has the functions of inducing plant disease resistance and plant stress resistance. Example 1 is the isolation and purification and structural identification of pullulan. Example 2 demonstrates that pullulan has significant antiviral effect on tobacco mosaic virus, and the main mode of action is protection; example 3 demonstrates that pullulan has significant induction of disease resistance activity against tobacco mosaic virus, and local use of pullulan can induce disease resistance in non-applied parts of tobacco; example 4 demonstrates that the expression level of the disease resistance gene in tobacco is improved after the pullulan treatment, which shows that the pullulan treatment induces the disease resistance of the tobacco; examples 5, 6 and 7 respectively prepare antiviral agents and fungicides containing pullulan, and the results show that the pullulan has good prevention and protection effects on tobacco mosaic virus, strawberry gray mold and cucumber powdery mildew, and show that the pullulan has broad spectrum on viral diseases and fungal diseases; chlorophyll content and leaf conductivity are important indexes for evaluating plant growth states, and examples 8 and 9 can obviously improve the chlorophyll content of the capsicum leaf and reduce the leaf conductivity after capsicum is treated by pullulan; example 10 demonstrates that pullulan can induce capsicum to resist low temperature stress. Examples 8, 9, 10 demonstrate that pullulan has an effect of inducing plant stress-tolerance activity and biostimulation. Those skilled in the art will appreciate that biological activity assays and physiological and biochemical experiments establish the general utility of pullulan as an elicitor, bactericide and biostimulant.

The pullulan of the present disclosure may be administered as a formulation comprising the pullulan by any of a variety of known techniques. For example, the compounds may be applied to the roots or leaves of plants to induce plant resistance or promote plant growth without compromising the commercial value of the plants. The pullulan may be administered in any of the commonly used formulation types, for example as a solution, powder, suspension, wettable powder, soluble liquid, flowable concentrate or emulsifiable concentrate, including in particular but not limited to: seed treatment emulsions, aqueous emulsions, macrogranules, microemulsions, water-soluble emulsions, water-dispersible granules, valleys, aerosols, bulk baits, slow-release blocks, concentrated baits, capsule granules, microcapsule suspensions, dry-mix seed powders, emulsifiable concentrates, electrostatic sprays, water-in-oil emulsions, oil-in-water emulsions, aerosol cans, fine granules, aerosol candles, aerosol cans, aerosol sticks, seed treatment suspensions, aerosol flakes, aerosol pellets, granular baits, hot aerosols, paint, microparticles, oil suspensions, oil-dispersible powders, flake baits, concentrated gels, pour-on agents, seed coatings, spread-on emulsions, film-forming oils, soluble powders, seed treatment water-soluble powders, ultra-low volume suspensions, tracking powders, ultra-low volume liquids, wet-mix water-dispersible powders.

Preferably, the pullulan of the present disclosure is administered in the form of a formulation comprising pullulan and a phytologically acceptable carrier. The concentrated formulation may be dispersed in water or other liquid for application, or the formulation may be dusty or granular. The formulations can be prepared according to procedures conventional in the agrochemical field. The present disclosure contemplates that all vehicles comprising pullulan for delivery and use as an elicitor can be formulated therewith, including all phytologically acceptable inert carriers, surfactants, emulsifiers, organic solvents or water, and the like.

The formulation may optionally include a combination containing other pesticides or other compounds having an anti-elicitation activity. Such additional pesticidal compounds or other compounds having an elicitor activity may be fungicides, insecticides, herbicides, nematicides, miticides, arthropodicides, bactericides, plant elicitors, or combinations thereof that are compatible with the pullulan and do not antagonize in the medium selected for application. Thus, in such embodiments, another pesticidal compound or an anti-elicitor active compound is used as a supplemental agent. The pullulan and another compound in combination can be generally combined in a ratio of 1:100 to 100:1 by weight.

Another embodiment of the invention is the use of pullulan in the preparation of biostimulants and their agricultural use. The essence is also to use the induction activity of pullulan.

Another embodiment of the invention is a method of applying pullulan for enhancing the immunocompetence of a plant and protecting the plant from infestation by pests, comprising applying pullulan to at least one of the plant, the area adjacent to the plant, soil suitable for supporting the growth of the plant, the root of the plant, and the foliage.

In order to better understand the essence of the invention, the technical contents of the invention will be described in detail with examples, but the invention is not limited to these examples.

Example 1: separation and structural identification of pullulan

10.0g of crude pullulan is diluted into 500mL of 2% aqueous solution, 250mL of chloroform/butanol mixed solution (volume ratio is 4:1) is added, the solution is fully oscillated to separate out protein, the solution is centrifuged for 30min at 4 000r/min, the precipitate is removed, and deproteinized supernatant is obtained. After the supernatant is dialyzed for 3d, the supernatant is concentrated to about 200mL under reduced pressure, 2 times of absolute ethyl alcohol is added, the mixture is fully stirred and left overnight, the precipitate is centrifuged for 30min at 8000r/min, and the precipitate is washed by acetone and diethyl ether in turn and then dried, thus obtaining 7.2g of crude pullulan powder. The deproteinized pullulan powder was dissolved in 30mL of distilled water, and subjected to DEAE-cellulose column chromatography at an elution rate of 1mL/min. Eluting with distilled water, collecting (5 mL per tube), colorimetrically identifying polysaccharide part by modified phenol-sulfuric acid method, collecting polysaccharide peak, concentrating under reduced pressure, dialyzing, and lyophilizing to obtain crude pullulan 5.6g. The crude pullulan is further subjected to Sephadex G-100 column chromatography, distilled water is used for eluting, the eluting speed is 4mL/h, the polysaccharide part is identified by colorimetric determination of a modified phenol-sulfuric acid method, and after collecting eluting peaks, the white powder is respectively frozen and dried to obtain 4.7G, and the total yield is 47.0%. The white powder obtained by the identification of nuclear magnetic resonance hydrogen spectrum (figure 1) and carbon spectrum (figure 2) is pure pullulan.

Example 2: determination of anti-TMV Activity of pullulan

(1) Protective Activity

Preparing pullulan polysaccharide solution with the concentration of 0.02mg/mL, 0.1mg/mL and 0.5mg/mL, selecting healthy 5-6 leaf stage heart leaf smoke with consistent growth vigor, spraying the medicament for 48 hours, inoculating TMV solution diluted 2000 times, treating with clear water as blank control, and treating with chitosan oligosaccharide with the positive control of 0.1 mg/mL. Each treatment was inoculated with 3 leaves, repeated 3 times, and after 3 days, the number of dead spots was counted, and the inhibition rate was calculated.

(2) In vitro passivation Activity

Pullulan solutions were prepared at concentrations of 0.02mg/mL, 0.1mg/mL and 0.5mg/mL and mixed with an equal volume of TMV solution diluted 1000-fold. And (3) placing for 1h at room temperature, and then inoculating the obtained product into healthy 5-6 leaf stage heart leaf cigarettes with consistent growth vigor. The blank control is clear water treatment, and the positive control is chitosan oligosaccharide with concentration of 0.1 mg/mL. Each treatment was inoculated with 3 leaves, repeated 3 times, and after 3 days, the number of dead spots was counted, and the inhibition rate was calculated.

(3) Therapeutic Activity

Preparing pullulan polysaccharide solution with the concentration of 0.02mg/mL, 0.1mg/mL and 0.5mg/mL, selecting healthy 5-6 leaf stage heart leaf cigarettes with consistent growth vigor, and spraying medicament for treatment after inoculating TMV solution diluted 2000 times for 48 hours. The blank control is clear water treatment, and the positive control is chitosan oligosaccharide with concentration of 0.1 mg/mL. Each treatment was inoculated with 3 leaves, repeated 3 times, and after 3 days, the number of dead spots was counted, and the inhibition rate was calculated.

Inhibition ratio (%) = (control number of dried spots-number of treated dried spots)/control number of dried spots×100

The results are shown in Table 1.

Table 1: protective, inactivating and therapeutic effects of pullulan on TMV

Note that: data are mean ± standard error, significant differences between 3 activities pass the Duncan Multiple Range Test (DMRT) in SPSS software, and different letters indicate significant differences between data at the 0.05 level.

From Table 1, the pullulan has good protection effect on TMV infected plants, which indicates that the tobacco has certain disease resistance effect by early application of the drug. The passivation effect of the pullulan on TMV is general, which indicates that the pullulan has no direct damage or weaker damage effect on TMV granules, and is inferior to the disease resistance effect caused by early administration.

Example 3: pullulan polysaccharide induced tobacco anti-TMV activity determination

And selecting the heart leaf tobacco for carrying out an induced disease resistance activity test, wherein the heart leaf tobacco can form virus dead spots, the symptoms on common tobacco are flowers and leaves, and different symptoms count disease resistance by different methods. The pullulan polysaccharide with the concentration of 0.02mg/mL, 0.1mg/mL and 0.5mg/mL is sprayed on the three lower leaves of the tobacco with the 6-7 leaf periods with consistent growth vigor. TMV was inoculated to the upper leaf after 48h without spraying. The blank control is clear water treatment, and the positive control is chitosan oligosaccharide solution. Each plant was inoculated with 2-3 leaves, each treatment included 10 tobacco plants, and the entire experiment was repeated 3 times. And after 3d, counting the disease index of the dead spot number of the tobacco leaf, wherein the formula of the calculated inhibition rate is as above, and the formula of the calculated prevention and treatment effect is as follows.

Disease index = Σ (disease number x disease number)/(highest disease number x total number of treatments) ×100;

control effect= (control disease refers to treatment disease refers to)/control disease refers to x 100%;

the results are shown in Table 2.

Table 2: induced disease resistance effect of pullulan on TMV

Note that: data are mean ± standard error, significant differences between 3 activities pass the Duncan Multiple Range Test (DMRT) in SPSS software, and different letters indicate significant differences between data at the 0.05 level.

As can be seen from table 2, the pullulan showed a remarkable effect of inducing plants to resist TMV on the tobacco leaves, demonstrating that the topical application of pullulan can induce disease resistance at the non-applied sites of tobacco.

Example 4: pullulan polysaccharide causes variation of tobacco disease resistance based on expression quantity

Spraying the 4-6 leaf stage tobacco with pullulan aqueous solution, and collecting samples 1 day after treatment. Extracting total RNA of tobacco by liquid nitrogen method, and determining the expression quantity change of the disease-resistant related genes NPR1, PR1 and PR2 by real-time fluorescence quantitative PCR.

As shown in figure 3, different concentrations of pullulan can cause obvious change of transcription level of PR protein of disease resistance related gene. At a concentration of 500mg/mL, the expression levels of NPR1, PR1, PR2 were increased 7.64,5.81,8.39-fold relative to the control group, respectively. Indicating that the pullulan induces disease-resistant defenses in tobacco bodies.

Example 5: prlulan polysaccharide preparation has effect of preventing tobacco mosaic virus in field district

The plot test is random arrangement, repeated for 3 times, plot area is equal to 60 square meters, the required fertility is uniform, the crop planting and management level is consistent, and protection rows are arranged between each treatment room and around the test area. And (3) carrying out foliar constant spraying, wherein 500 times of the liquid of the altaic is used as a control medicament, the foliar constant spraying is used as a positive medicament control, and the water control is arranged. All reagents must be diluted twice. Spraying medicine from the leaf stage of the heart leaf tobacco for 4-5 times, and spraying for 1 time every 4 days, wherein the total time is 3 times. Taking the whole leaf on the top after 2d of the last spraying, rubbing and inoculating TMV, inoculating 3 leaves for each plant, repeating the process for three times for 10 plants each time, investigating the disease index of each treatment after 10d of virus inoculation, and calculating the control effect.

Disease grading standard is according to the tobacco industry standard of the people's republic of China-tobacco mosaic virus severity grading investigation method (YC/T39-1996):

level 0: the whole plant is free from diseases;

stage 1: heart She Maiming or slight leaves, or upper 1/3 leaves, but not deformed, the plants are not obviously dwarfed;

2 stages: 1/3 to 1/2 of the leaves of the leaf flower, or a few of the leaves are deformed; or the main vein is black, and the plant dwarfs more than 2/3 of the normal plant height;

3 stages: when flowers are 1/2 to 2/3, or deformation or main side vein is blackened, the plant is dwarfed to be 1/2 to 2/3 of the normal plant height;

4 stages: the whole plant leaves and flowers are severely malformed or necrotic, and the sick plant is dwarfed to be the normal plant height of l/3 to 1/2. To refine the survey results, based on the above severity classification, the classification standard is refined, the l+ level is added between the 1 and 2 levels, the 2+ level is added between the 2 and 3 levels, the 3+ level is added between the 3 and 4 levels, and the levels are recorded as 1.5, 2.5 and 3.5:

1+ stage: heart She Maiming or slight leaves, or 1/3 leaf leaves at the upper part to slightly shrink, the plants have no obvious dwarfing;

2+ stage: l/3 to 1/2 of leaves, flower leaves and leaves are deformed or main veins are blackened, and the plant is dwarfed to be more than 2/3 of that of a normal plant;

3+ stage: 1/2 to 2/3 of the leaves of the leaf flowers, or the deformation or main vein necrosis, or the dwarfing of the plant to 1/2 of the normal plant height.

And calculating the disease index according to the severity, wherein the prevention and treatment effects are used as the measurement of the effects of different treatments.

Disease index = Σ [ (number of infected plants×severity grade representative value)/(total investigation number×severity highest grade representative value) ]100%

Control% = ((control mean disease index-treatment mean disease index)/control mean disease index) ×100%

The results are shown in Table 3.

Table 3: field district drug efficacy test of pullulan preparation for preventing and treating tobacco virus diseases

Test agent	Dilution factor/fold	Preventing effect (%)
			Pullulan polysaccharide soluble liquid (30% content)	200	66.06
Pullulan polysaccharide soluble liquid (1% content)	200	38.88
			Pullulan polysaccharide soluble liquid (80% content)	200	76.15
Clear water control	-	0

From the table above, the pullulan preparation has good prevention and control effects on tobacco mosaic virus.

Example 6: field plot pharmacodynamic test of pullulan preparation on strawberry gray mold

And selecting greenhouse strawberries for cell tests. The greenhouse test land is generally uniform in fertility, consistent in planting level and illThe occurrence and hazard degree are relatively uniform, and the control and management are convenient. Protective rows are arranged between the treatments and around the test area, the area is 60 square meters, and the test is repeated for 3 times. The liquid consumption is 10kg/60m ² . And (3) respectively using clear water and 500 times of the liquid of the altaic as negative and positive controls to carry out foliar spray. All reagents must be diluted twice. Spraying medicine from strawberry to 2 months, sealing, spraying medicine 1 time every 5d, and 3 times. And (5) investigation of disease leaf morbidity and statistics of disease index after the last spraying, and calculation of control effect. The disease classification criteria were as follows:

level 0: no disease spots;

stage 1: the area of the disease spots is below 5%;

3 stages: 6% -10% of the area of the disease spots;

5 stages: the area of the lesion is 11% -20%;

7 stages: 21% -50% of the area of the lesion;

stage 9: the area of the disease spots is more than 50 percent.

The disease index and the prevention and treatment effect are calculated by the following formulas.

Disease index = Σ100% (number of leaves per stage x relative number of stages)/(number of total investigation × 9) ]%

Control% = [ (control mean disease index-treatment mean disease index)/control mean disease index ] ×100%

The results are shown in Table 4.

Table 4: pullulan polysaccharide preparation and field efficacy test for preventing and treating strawberry gray mold by using preparation containing essential oil and extract of pullulan polysaccharide

Test agent	Dilution factor/fold	Preventing effect (%)
			Pullulan (Prlulan)Polysaccharide soluble liquid (30% content)	200	58.02
Pullulan polysaccharide soluble liquid (1% content)	200	22.3
			Pullulan polysaccharide soluble liquid (80% content)	200	75.9
Clear water control	-	0

From the table, the pullulan preparation has good prevention and protection effects on the gray mold of strawberries.

Example 7: field plot pharmacodynamic test of pullulan preparation on cucumber powdery mildew

The plot test is random arrangement, repeated for 3 times, plot area is equal to 60 square meters, the required fertility is uniform, the crop planting and management level is consistent, and protection rows are arranged between each treatment room and around the test area. And (3) spraying the leaf surface with a constant amount, and respectively taking clear water and 500 times of the liquid of the atenolol as negative and positive controls to spray the leaf surface. All reagents must be diluted twice. Spraying the pesticide when 5-6 true leaves are planted after the cucumber is planted, wherein the spraying amount is uniform to spray the leaf surface, and the pesticide liquid begins to drop. Spraying 1 time every 7d for 3 times. And (5) investigation of disease indexes after 15d of the last spraying, and calculation of the control effect. 5 plants are randomly selected from each cell for investigation, 5 leaves are respectively investigated according to the upper part, the middle part and the lower part of each plant. The disease classification criteria were as follows:

level 0: no disease spots;

stage 1: the area of the disease spots accounts for less than 5% of the whole leaf area;

3 stages: the area of the lesion accounts for 6-10% of the whole leaf area;

5 stages: the area of the lesion accounts for 11% -25% of the whole leaf area;

7 stages: the area of the lesion accounts for 26% -50% of the whole leaf area;

stage 9: the area of the disease spots accounts for more than 50% of the whole leaf area.

Disease index = Σ100% (number of leaves per stage x relative number of stages)/(number of total investigation × 9) ]%

Control% = ((control mean disease index-treatment mean disease index)/control mean disease index) ×100%

The results are shown in Table 5.

Table 5: field control effect of pullulan preparation for controlling cucumber powdery mildew

Test agent	Dilution factor/fold	Preventing effect (%)
			Pullulan polysaccharide soluble liquid (30% content)	200	44.1
Pullulan polysaccharide soluble liquid (1% content)	200	18.16
			Pullulan polysaccharide soluble liquid (80% content)	200	63.62
Clear water control	-	0

From the above table, the pullulan preparation has good prevention effect on powdery mildew of cucumber.

Example 8: effect of pullulan treatment on chlorophyll content of capsicum

10 capsicum plants with consistent growth vigor are randomly selected in each district, the functional leaves on the upper part are picked for measurement, and each treatment is repeated three times. Fresh and wiped pepper leaves are taken, the middling veins are removed, sheared and evenly mixed for measurement. And calculating the concentration of chlorophyll a and chlorophyll b of the pepper leaves according to the formula, and converting the concentration into the total chlorophyll mass in the leaves.

C _a+b ＝C _a +C _b ＝8.05OD ₆₆₃ +20.29OD ₆₄₅

The results are shown in Table 6.

Table 6: effect of pullulan treatment on chlorophyll content

As can be seen from the table above, different concentrations of pullulan treatment had a certain effect on chlorophyll content, wherein the chlorophyll content was increased by 7.05%, 16.4% and 22.82% under the treatment of pullulan at 0.02mg/mL, 0.1mg/mL and 0.5mg/mL, respectively, compared with the blank. Therefore, the pullulan treatment can obviously improve the chlorophyll content in the capsicum leaves, thereby promoting the growth of plants.

Example 9: determination of the influence of pullulan on the relative permeability of Capsici fructus cell membranes

The stress resistance activity of the plant after the medicament treatment is indirectly reflected by measuring the membrane permeability of the leaf cell. Cleaning the blade, and punching by a puncher. 0.3g of perforated blade is weighed into a clean 100mL beaker, rinsed 3 times with 80mL of deionized water, added with 50mL of deionized water, left to stand for 3 hours, and the conductivity is measured by a conductivity meter. The conductivity was measured immediately after cooling in a boiling water bath for 15min after measurement. The calculation is as follows: conductivity (%) = (treatment conductivity/boiling conductivity) ×100

The results are shown in Table 7.

Table 7: effect of pullulan treatment on conductivity of capsicum leaves

From the above, the conductivity of the leaves treated by pullulan decreases with the increase of concentration, so that the adaptability of the plants to the environment is improved.

Example 10: effect of pullulan on low temperature stress resistance of capsicum

Sterilizing pepper seeds with 5% sodium hypochlorite solution, soaking in clear water for 24 hr, wrapping with gauze, placing in incubator (30deg.C dark constant temperature) for germination acceleration, and repeatedly washing with sterile water every 8 hr. After the seeds are exposed to white, the seeds are sown in seedling raising plug trays (32 holes) and placed in a greenhouse (RH=70% -80%, T=25+ -5 ℃) for continuous culture. When the pepper seedlings grow to 5-6 true leaves, selecting the seedlings with consistent growth for testing. Different low temperature treatments are set up for moderate low temperature stress in a climatic incubator and under fixed culture conditions (rh=60% -70%; L: d=16 h/8h, illumination intensity: 4000 lxs): 12.5 ℃ (16 h L)/7.5 ℃ (8 h D). The pepper is placed in a low-temperature stress environment and then is applied 1 time every 5 days, the application concentration is 0.5mg/mL, the morphological index is measured after 15 days, and the strong seedling index is calculated according to the following formula: strong seedling index= (stem thickness/plant height + root dry mass/aerial part dry mass) x whole plant dry mass. As a blank, the clear water control was used, and 9 replicates were treated each.

TABLE 8 Pullulan polysaccharide (0.5 mg/mL) treatment induced Capsici fructus to resist Low temperature stress

Treatment of	Height of plant (cm)	Stem thickness (mm)	Root-to-crown ratio	Index of strong seedlings
					Treatment group	12.37±0.12	2.28±0.03	0.23±0.01	0.38±0.02
Pullulan polysaccharide	12.98±0.17	2.34±0.01	0.24±0.02	0.45±0.02

As can be seen from table 8, the growth condition of the capsicum treated with pullulan is better than that of the blank control under the condition of low temperature stress, which indicates that the pullulan has the activity of inducing capsicum to resist low temperature stress.

The preferred embodiments of the present disclosure have been described in detail above with reference to the accompanying drawings, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.

In addition, the specific features described in the foregoing embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present disclosure does not further describe various possible combinations.

Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.

Claims (1)

1. The application of the pullulan polysaccharide in preparing a plant resistance inducer has the application range of inducing tobacco to resist tobacco mosaic virus;

the pullulan is linear polysaccharide formed by polymerizing a maltotriose repeating unit connected with alpha-1, 4 glycosidic bonds through alpha-1, 6 glycosidic bonds, and the molecular weight is 2-200 ten thousand daltons;

the content of the pullulan is 0.02-0.5 mg/mL.