CN108056103B

CN108056103B - Glucosaccharide compound preparation and application thereof in potato disease prevention and control

Info

Publication number: CN108056103B
Application number: CN201610978608.0A
Authority: CN
Inventors: 尹恒; 王文霞; 赵小明; 李悝悝
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2016-11-08
Filing date: 2016-11-08
Publication date: 2020-09-22
Anticipated expiration: 2036-11-08
Also published as: CN108056103A

Abstract

The invention relates to a compound preparation of glucooligosaccharide, which contains the glucooligosaccharide and a botanical fungicide. The weight percentage of the gluco-oligosaccharide in the compound preparation is 0.025 percent to 0.1 percent. The botanical fungicide accounts for 0.05-0.1% of the weight of the compound preparation. The compound preparation can be used for preventing and treating potato late blight, can be used for dressing seed potatoes before potato planting, can be used for multiple times in seedling stage and tuber formation stage, can effectively prevent and treat potato late blight, can slow down the generation of drug resistance of germs, has strong controllability and small risk on the generation of diseases, and has good development and application prospects.

Description

Glucosaccharide compound preparation and application thereof in potato disease prevention and control

Technical Field

The invention relates to the field of potato disease control, in particular to a glucooligosaccharide compound preparation for controlling potato late blight and an application method thereof.

Background

Potatoes are the fourth crop in the world, and have gradually become world-wide agricultural resources and various industrial processing raw materials due to the characteristics of high yield, short growth period, strong adaptability, rich nutrition, wide application and the like. China is the first major country for potato planting, and the production area and the yield both account for about one fourth of the world. According to data of Ministry of agriculture, the potato planting area of China reaches 5570 kilo hectares (8355 ten thousand mu) in 2014. The potato is often damaged by plant diseases and insect pests in the growth process, and the yield and the quality are seriously influenced. The potato late blight is one of the main diseases, is a destructive disease, has high epidemic speed and wide damage range, and can cause death of potato stems and leaves and rot of tubers. At present, the disease occurs and prevails in all main potato production areas in the world, the harmfulness, the control difficulty and the influence on the society exceed rice blast and wheat rust, the disease becomes the first crop disease in the world, and according to statistics, the economic loss (direct loss and medicament cost) caused by late blight in the world every year reaches $ 30-50 hundred million. At present, because the main-planted potato variety is mostly not resistant to late blight, a grower mainly adopts a chemical method to prevent and treat the disease. However, long-term use of chemical pesticides causes environmental pollution, pesticide residues, and also causes problems such as generation of drug-resistant strains and cross-resistance. In addition, the chemical prevention and control of the potato late blight need to grasp a critical period, but the medication time is difficult to grasp, and the disease prevalence and the serious loss are caused if the medication is not timely.

The beta-glucosaccharase can induce various plants to generate resistance, and the reported plants comprise tobacco, arabidopsis thaliana, rice, grapes and the like, but the induction effect of the beta-glucosaccharase with different structures on different plants is very different, for example, the beta-1, 3 beta-1, 6 glucoheptaose with a branch structure can induce soybeans to generate phytoalexin, while the linear beta-1, 6-glucosaccharase does not have the induction effect. The linear beta-1, 3 gluco-oligosaccharide can induce the resistance of tobacco and tomato, but has no obvious effect on parsley. Currently, beta-glucan oligosaccharides derived from kelp have been developed as biopesticides and have been approved by the U.S. environmental protection agency. However, kelp grown in the sea area of China has a low content of kelp starch, which is only 0.5 to 1% of the dry weight, compared with the European palmate kelp, and meanwhile, the relatively pure kelp starch is expensive, and the development of the kelp starch as a biopesticide has disadvantages of raw materials and price. The curdlan polysaccharide is water-insoluble glucan, is a linear molecule formed by connecting glucose residues through beta-1, 3 glycosidic bonds, is produced by Agrobacterium sp.

Recent studies have reported that curdlan oligosaccharides can induce the production of defense responses in potato leaf cells. And the potato leaves are treated by curdlan oligosaccharide 1d before P.infestans infection, the damage ratio of the leaves is reduced to 7.79% + -3.03% after 2 weeks, the damage area ratio of the control leaves reaches 15.82% + -5.44%, the treatment prevention effect of the curdlan oligosaccharide reaches 50%, however, compared with the drug effect of the conventional chemical agent, the difference is large, and the actual field application degree cannot be reached.

Aiming at the technical problems of the chemical prevention and control of the potato late blight and the effects and the defects of the curdlan oligosaccharide on the prevention and the control of the potato late blight, the invention aims to obtain a compound preparation capable of effectively preventing and controlling the potato late blight by compounding the curdlan oligosaccharide and a botanical fungicide, realize the biological prevention and the control of the potato late blight and overcome the defects of the existing chemical prevention and control technology of the potato late blight.

At present, the research, development and application of the curdlan oligosaccharide and plant bactericide compound preparation for preventing and treating the potato late blight are not reported in documents and patents. Both the curdlan oligosaccharide and the botanical fungicide are derived from natural products, have the characteristics of safety, environmental protection, low residue and the like, are used for preventing and treating the potato late blight, and have advantages compared with other means. The invention aims to provide a biological prevention and control technology which meets the environmental protection requirement and can effectively prevent and control the potato late blight based on the prevention and control thought of green environmental protection and prevention.

The invention content is as follows:

the invention aims to provide a glucooligosaccharide compound preparation and application thereof in preventing and treating potato late blight.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the compound preparation of the glucooligosaccharide is a liquid preparation formed by mixing the glucooligosaccharide and a botanical fungicide. Wherein the concentration of the gluco-oligosaccharide in the compound preparation is 250-1000mg/L, and the concentration of the botanical fungicide in the compound preparation is 500-1000 mg/L.

The gluco-oligosaccharide in the compound preparation is curdlan oligosaccharide prepared by enzymolysis, and the average molecular weight is 500-5000 Da.

The botanical fungicide contained in the compound preparation is one or more than two of matrine, eugenol, aloperine and wilfordine.

The compound preparation can be used for preventing and treating potato late blight, and any agriculturally available dosage form is prepared from the composition containing the glucooligosaccharide, the botanical fungicide and appropriate auxiliary materials according to factors such as prevention and treatment objects, environmental conditions, prevention and treatment methods, prevention and treatment cost and the like; the preferable dosage forms are suspending agents and water dispersible granules.

The specific method for preventing and treating the potato late blight by using the compound preparation comprises the step of dressing seeds of potato seeds by using the compound preparation before planting, wherein the dosage of the seed potato seeds is 1-2L/100kg of seed potato blocks. Then spraying for 2-3 times in seedling stage and tuber forming stage, wherein the spraying dosage is 30-50L/mu each time.

The invention has the advantages and beneficial effects that:

(1) the compound preparation of the glucooligosaccharide comprehensively considers two factors of plant induced resistance and bacteriostasis. Inducing resistance of the potatoes through treatment of the gluco-oligosaccharides; through the treatment of the bactericide, the growth and the reproduction of microorganisms are inhibited, a more ideal control effect is comprehensively achieved, and in the carried out embodiment, the effect is better than the treatment effect of the existing commercial reagent.

(2) The curdlan oligosaccharide used in the compound preparation provided by the invention belongs to natural products, and the bactericide used in the compound preparation belongs to a plant source, so that the compound preparation has no pollution to the environment and no health hazard to human bodies when being used for preventing and treating potato late blight. Compared with the method of using the bactericide only, the usage amount of the bactericide is greatly reduced.

(3) The compound preparation provided by the invention can be used for multiple times in the sowing period, the seedling period, the tuber forming period and the harvesting period of potato seeds. Has the advantages of simple application method, low cost and easy popularization and application. Compared with the prior art, the prevention is mainly carried out, and the plant source bactericide is used for assisting, so that the remarkable synergistic effect can be obtained by starting from two aspects of improving the disease resistance of plants and reducing the quantity of pathogenic bacteria, the defect of single function of the oligosaccharide resistance inducer is overcome, the using amount of chemical pesticides is greatly reduced, the pesticide application effect is improved, the pesticide cost is reduced, the generation of drug resistance is slowed down, the controllability on the generation of diseases is strong, the risk is small, and the development and application prospect is good.

Drawings

FIG. 1 is a statistical chart of the process of producing hydrogen peroxide by using different concentrations of glucooligosaccharides to induce potato suspension cells;

FIG. 2 is a graph showing that gluco-oligosaccharide (500mg/L) induces the expression of alkaline glucanase in potato suspension cells;

FIG. 3 is a graph showing that gluco-oligosaccharide (500mg/L) induces the expression of alkaline chitinase in potato suspension cells.

Detailed Description

The invention is further illustrated by the following examples:

example 1: experiment for inducing potato tuber to resist late blight by using glucooligosaccharide compound preparation

Fresh and healthy potato tubers are taken, cleaned and soaked in 2% NaClO for 10min for surface disinfection, then washed with sterile water to remove residual NaClO, cut into small blocks of 2.0cm multiplied by 0.5cm by a sterile cutter, cleaned with sterile deionized water for 3 times, and subjected to residual water absorption by sterilized filter paper. Placing 10mlL Glucosan oligosaccharide compound preparation in sterilized 9cm petri dish, and soaking one end of small potato piece about 2mm (induction end) for 30 min. While sterile water-treated potato tubers were used as controls, 6 in parallel per group. Then, the residual excitons on the surface of the tubers are washed clean by sterile water, and the residual moisture on the surface is sucked dry by sterilized filter paper. Placing in a sterilized culture dish paved with filter paper, and culturing for 24h in a constant temperature incubator at 20 ℃ in dark and with moisture preservation.

Taking the phytophthora infestans sporangium suspension to dilute to 20-30 sporangiums in each visual field under a 10-fold magnifying glass. And (3) inoculating 20uL of the seed to the center of the non-induction end of the potato tuber after 24h of treatment of the compound preparation. And (5) performing moisture preservation culture in a 20-DEG C constant-temperature incubator. After 7 days, the induced resistance effect is observed, and the disease index is calculated.

The disease index grading standard is as follows:

level 0: growth of sterile filaments on tubers;

level 1: mycelia are visible on tubers, and no tissue degradation occurs;

and 2, stage: the mycelium on the tuber covers half of the area of the tuber, and tissue degradation does not occur;

3, grade; the mycelium covered the entire tuber with no or slight tissue degradation;

4, level: the mycelium covered the entire tuber and the tissue degradation was severe.

Disease index ∑ (disease grade number × disease grade block number)/survey total number × highest disease grade number) × 100%

The induced resistance effect is (contrast disease index-treatment disease index)/contrast disease index is multiplied by 100 percent

TABLE 1 specific formulation and control effect of compound preparation of gluco-oligosaccharide and matrine

TABLE 2 specific formulation and control effect of the compound preparation of gluco-oligosaccharide and eugenol

Table 3. specific formulation and control effect of the formulation of glucooligosaccharides and aloperine:

compound preparation		Disease index%	Induced resistance effect%
				Glucosaccharide 250mg/L	Soaloperine 500mg/L	24.2	71.53
	Soaloperine 750mg/L	21.8	74.35
					Soaloperine 1000mg/L	24.3	71.41
Glucosaccharide 500mg/L	Soaloperine 500mg/L	21.5	74.71
					Soaloperine 750mg/L	22.6	73.41
	Soaloperine 1000mg/L	26.3	69.06
				Glucosaccharide 250mg/L		58.7	30.94
Glucosaccharide 500mg/L		39.2	53.88
					Soaloperine 500mg/L	49.0	42.35
	Soaloperine 750mg/L	44.5	47.65
					Soaloperine 1000mg/L	42.6	49.88
Clear water control		85.0	0.00

TABLE 4 specific formulation and prevention and treatment effect of compound preparation of glucooligosaccharides and triptolide

The test results of the glucan oligosaccharide compound preparation induced potato tubers to resist late blight are shown in tables 1-4, the disease index of a clear water control group is more than 85%, the control effect of the glucan oligosaccharide alone induced treatment on the potato tubers to the late blight is about 50%, the control effect is remarkably increased after the glucan oligosaccharide compound preparation induced treatment, the control effect is up to 83%, and the compound preparation has good capability of inducing the potato tubers to resist the late blight infection.

Example 2 Glucosaccharide Complex formulation Induction of Potato plants against Phytophthora infestans

Washing the surfaces of leaves of potato plants (7-8 weeks) with deionized water, naturally airing, and inducing the leaf surfaces of the potato plants with a glucooligosaccharide compound preparation, wherein 3 plants are treated with each inducer to serve as a parallel experiment. After induction 24, the concentration is 10⁵Potato plants were sprayed with a/mL suspension of p.infestans spores. The resistance inducing effect is evaluated by adopting the proportion calculation of the damaged area of the leaves, and each treatment group samples 30 leaves in total. After 2 weeks of infecting potato leaves with the p.infestans spore suspension, the area of the damaged leaves was counted, and the specific results are shown in the following table. The ratio of the damaged area of the blade in the clear water control group reaches 25.31% +/-3.44% after 2 weeks, and the damaged area of the blade is greatly reduced after 2 weeks by treating the blade with the compound preparation of the gluco-oligosaccharide 1d before P.infestans infection, and the prevention effect is 78% -91%.

TABLE 5 Glucosaccharide complex formulation induced potato plants against Phytophthora infestans

Treatment of	The ratio of damaged area of leaves%	Prevention effect
			Clear water control	25.31±3.44	—
250ppm of gluco-oligosaccharide and 750ppm of matrine	4.89±1.11	80.68
			500ppm of gluco-oligosaccharide and 750ppm of matrine	3.77±1.65	85.10
250ppm glucooligosaccharides +750ppm eugenol	2.83±1.31	88.82
			500ppm Glucosaccharide +750ppm eugenol	2.18±1.17	91.39
250ppm of glucooligosaccharides +750ppm of aloperine	3.34±1.02	86.80
			500ppm Glucosaccharide +750ppm Sophora alopecuroide	3.03±1.27	88.03
250ppm glucooligosaccharides +750ppm triptolide	5.35±2.88	78.86
			500ppm Glucosaccharide +750ppm Tripterygium wilfordii alkaloid	4.01±3.15	84.16
250ppm Glucosaccharide	13.90±3.03	45.08
			500ppm Glucosaccharide	12.89±2.75	49.07
750ppm matrine	19.92±3.25	21.30
			750ppm eugenol	19.48±2.64	23.03
750ppm Sophora alopecuroide alkali	18.97±3.37	25.05
			750ppm Tripterygium wilfordii alkaloid	20.20±2.91	20.19

Example 3 experiment of Glucosaccharide compounded preparation for preventing and treating potato late blight in residential area

The test field is arranged in the Yanggang area of the Xiyangyang city of Shaanxi province, and the potato variety is No. 9 sunstroke. The potato sowing date is 5 months in 2014, the sowing density is 3500 strains/mu, the ridge spacing is 50cm, the plant spacing is 30cm, and the potato is cut into pieces and sowed. When the potato is planted, a high-concentration special fertilizer for potatoes is applied, and the cultivation conditions and management measures of all test districts are consistent.

The experiment shows that 8 compound preparations are treated, specifically shown in the following table, and a blank control of spraying clear water is additionally set, wherein the blank control is 75 percentMancozeb is a positive control. Each treatment for 3 repetitions, adopting random block design, total 30 cells, each cell area 30m²Total area 900m². Isolation zones of 0.6m width were provided between cells and around the test site.

And (3) seed potato treatment, namely soaking the seed potatoes into the treatment preparation for 30min before sowing, stacking the seed potatoes in a dry place for naturally drying, and cutting more than 50g of seed potatoes into 20-30g of potato blocks before sowing, wherein each potato block is provided with a bud eye.

Treating seedlings, namely spraying the treatment preparation to the seedlings and the soil around the seedlings after the length of the seedlings growing from the bud eyes is 5-10cm, and spraying 0.6L of the preparation to each 10 square meters of field;

and (3) treating in a tuber formation period, wherein the treatment preparation is sprayed to the plants and soil around the plants in the early tuber formation period (bud period) and the full tuber formation period (flowering period), and 0.6L of the preparation is sprayed to each 10 square meters of the field.

Disease conditions were investigated 7 days after the last application, 5 sampling methods were used in each plot, 3 plants were investigated in each plot, 10 leaves were investigated in the lower, middle and upper parts of each plant, and the grade of the diseased leaves was recorded. Grading standard: grade 0, no symptoms;

grade 1, with individual lesions on the leaves

Grade 3, 1/3 leaf with scabs

Grade 5, there are spots on 1/3-1/2 leaves

Grade 7, 1/2-2/3 leaf blade infection

Susceptibility of blade of 9 th grade and above 2/3-

Calculating disease index and disease index control effect, and performing difference analysis on the control effect data processed differently by adopting SPS version software.

Disease index [ Σ (number of onset stages × number of representative stages)/(total number of leaves investigated × 9) ] × 100

Preventing and treating effect (%) - (blank control area disease index-medicament treatment area disease index)/blank control area disease index multiplied by 100

The yield measuring method in the harvest period comprises the following steps: and (3) continuously measuring 20 plants in different regions for yield measurement after eliminating the marginal effect in each cell, repeating the measurement for 3 times, dividing the potato blocks into 2 grades of large potatoes and small potatoes, recording the grades, wherein the small potatoes are smaller than 100g, the large potatoes are larger than or equal to 100g, and calculating the yield, the yield increase and the yield increase rate of the large potatoes. Meanwhile, the rotten potato rate is counted

TABLE 6 field control of the Glucosaccharide combination against potato late blight

Treatment of	Disease index%	Control effect%
			Clear water control	44.59
500ppm of gluco-oligosaccharide and 750ppm of matrine	3.45	92.26
			500ppm Glucosaccharide +750ppm eugenol	2.87	94.46
500ppm Glucosaccharide +750ppm Sophora alopecuroide	3.05	93.16
			500ppm Glucosaccharide +750ppm Tripterygium wilfordii alkaloid	3.68	91.75
500ppm Glucosaccharide	23.75	46.74
			750ppm matrine	22.52	49.50
750ppm eugenol	10.74	75.91
			750ppm Sophora alopecuroide alkali	12.15	72.75
750ppm Tripterygium wilfordii alkaloid	13.64	69.41
			75% mancozeb 800 times diluted	8.85	80.15

TABLE 7 Effect of Glucosaccharide complex formulation on the prevention and treatment of potato late blight on yield

The investigation results are shown in tables 6-7, and field plot experiments show that the prevention effect of the glucooligosaccharide compound preparation on potato late blight is more than 90 percent and is 75 percent higher than that of mancozeb which is a commonly used prevention and control agent in production. The rotten potato rate of the potatoes treated by the gluco-oligosaccharide compound preparation is greatly reduced, and the yield is remarkably improved.

Example 4 Effect of Glucosaccharides on inducing the production of defense responses in Potato leaf cells

The specific fluorescent probe 2',7' -dichlorodihydrofluorescein diethyl ester of hydrogen peroxide is applied, potato suspension cells are used as experimental materials, the content of hydrogen peroxide in the cells after treatment of the glucooligosaccharides with different concentrations is detected by a fluorescence microplate reader, and the determination result is shown in the following figure, wherein the glucooligosaccharides with the concentrations of 250ppm to 1000ppm can obviously induce the generation of the hydrogen peroxide. Meanwhile, the gluco-oligosaccharide can also induce the expression of the protein related to the pathogenesis of the potato suspension cells, wherein the glucanase is remarkably increased within 48-96h after treatment, and the experimental results show that the gluco-oligosaccharide can activate the defense reaction of the potato suspension cells.

Claims

1. A compound preparation of glucooligosaccharides is characterized in that: the compound preparation is a liquid preparation formed by mixing gluco-oligosaccharide and a botanical fungicide, wherein the gluco-oligosaccharide is curdlan oligosaccharide prepared by enzymolysis, the average molecular weight is 500-5000Da, the concentration of the gluco-oligosaccharide in the compound preparation is 250-1000mg/L, and the concentration of the botanical fungicide in the compound preparation is 500-1000 mg/L, and the botanical fungicide is one of matrine, eugenol, aloperine and wilfordine.

2. The use of the compounded preparation of claim 1 in the control of potato diseases.

3. The use of the compounded preparation according to claim 2, characterized in that: the potato disease control is potato late blight control.

4. The use of the compounded preparation according to claim 2, characterized in that: the compound preparation containing the gluco-oligosaccharide and the botanical fungicide and proper auxiliary materials are prepared into the suspending agent which can be used in agriculture.

5. The use of the compounded preparation according to claim 3, characterized in that: the method for preventing and treating the potato late blight comprises the steps of dressing seeds of potato seeds with the compound preparation before planting; then spraying for 2-3 times in seedling stage and tuber forming stage.

6. The use of the compounded preparation according to claim 5, characterized in that: before planting, dressing seed potato with the compound preparation, wherein the dosage is 1-2L/100kg seed potato cut; then the spraying dosage is 30-50L/mu each time in the seedling stage and the tuber forming stage.