Patent Description:
Effective autoimmune systems have been formed in plants in response to invasions of pathogenic bacteria through long-term evolution and development. Plant resistance inducers can stimulate immune systems of plants to resist, prevent and treat diseases. With respect to interactions between plants and pathogens, on one hand, plants secrete β-<NUM>,<NUM> glucanase, chitosanase and chitinase to directly hydrolyze cell walls of pathogenic bacteria and inhibit their growth; on the other hand, pathogenic bacteria secrete polygalacturonase and pectinase to degrade cell walls of plants. Oligosaccharide fragments produced through the mutual hydrolyses can stimulate plants to produce pathogenesis-related proteins, phytoalexins, etc., thereby enhancing plant disease resistance. This is the basic principle of oligosaccharides as plant disease resistance inducers.

At present, research on the mechanism of oligosaccharides has developed from general observation to molecular and cellular levels. A large number of studies reveal that oligosaccharides play a role of elicitors generally in the following manner. Mutual recognition of oligosaccharides and receptors on cell membranes causes changes in receptor conformation and produces transmembrane signals; through a series of signal transduction, amplification and integration, expression of defense genes is regulated, secondary metabolites are accumulated, and plants are induced to produce autoimmune resistance to infection of pathogenic substances.

Oligosaccharide elicitors are derived from natural products and have the following functional characteristics according to the current research. (<NUM>) They are ecologically-friendly. (<NUM>) They are effective against pathogenic bacteria that are ineffectively prevented and treated by conventional methods, especially those resistant to chemical pesticides. (<NUM>) They act on plants rather than directly on pathogenic organisms, which avoids adverse effects on non-pathogenic organisms. (<NUM>) They protect plants from various biotic stresses of microorganisms, insects, nematodes, etc. (<NUM>) They can be combined with other prevention and treatment methods acting in different ways to expand the scope of prevention and treatment. (<NUM>) They activate various genes for systemic resistance, some of which may protect plants from high and low temperatures, drought and ultraviolet stresses.

Oligosaccharide elicitors that have been extensively studied in recent years mainly include the following types. (<NUM>) Chitosan oligosaccharides: oligosaccharide products with a degree of polymerization (DP) between <NUM> and <NUM> obtained through the degradation of chitosan by means of a special biological enzyme technology; can induce plant disease resistance, elicit immunity against and kill various fungi, bacteria and viruses; have favorable prevention and treatment effects on cotton verticillium wilt, rice blast, tomato late blight and other diseases; and can be developed into biological pesticides, growth regulators, fertilizers, etc. (<NUM>) Chitin oligosaccharides: degradation products of chitin, are recognized as a general term of saccharides composed of <NUM>-<NUM> N-acetylglucosamines linked by glycosidic bonds, and have a good inhibitory effect on various pathogenic bacteria in plants, such as those causing wheat sharp eyespot and tobacco black shank. (<NUM>) Glucooligosaccharides: a series of oligosaccharide elicitors that people studied at first and knew systematically; can effectively induce the synthesis and accumulation of phytoalexins; serve as early informational molecules that are of great significance for plants in: the resistance to diseases and infection, molecular signal regulation, growth and development, morphogenesis, adaptation to the environment and other aspects; and have favorable prevention and treatment effects on wheat powdery mildew, potato late blight, etc. (<NUM>) Oligogalacturonic acids: oligosaccharides composed of <NUM>-<NUM> galacturonic acids linked by α-<NUM>,<NUM>-glycosidic bonds, derived from pectic polysaccharides and widely distributed in primary cell walls and intercellular layers of roots, stems, leaves and fruits of higher plants; play a role in softening and adhesion of cell tissues; and have favorable prevention and treatment effects on pepper virus and apple mosaic virus diseases.

Low-DP β-<NUM>,<NUM>-glucooligosaccharides prepared from laminarin have been proved to have a favorable disease prevention effect on various plants, and relevant products have been marketed. However, oligosaccharides produced from laminarin have a low DP and are not uniform due to limitations of sources of raw materials and the hydrolysis process, making it difficult to give full play to their functions. Therefore, there is an urgent need to find an ideal alternative raw material to efficiently prepare oligosaccharides with a high DP and a uniform structure, and to explore the plant induction mechanism of new oligosaccharides.

<NPL> describes antitumor effects of riclin being a succinoglycan. <NPL>, XP005295703 describes <NUM>H and <NUM>C-nuclear magnetic resonance assignments for determination of locations of the O-succinyl and O-acetyl substituents. <NPL> describes Agrobacterium sp. ZCC3656 being a highly stable EPS-producing strain. FU <NPL>, (<NUM>), XP037582744 describes a homogeneous octaose which was depolymerized from the succinoglycan Riclin and was investigated as a novel elicitor to activate the immune system of potato (Solanum tuberosum L.

The present invention aims to provide an application of a heteropoly oligosaccharide in improving disease resistance of a plant.

The heteropoly oligosaccharide contains seven D-glucose residues and one D-galactose residue, and the structure thereof is represented as follows:
<CHM>
R<NUM> is H or a monomolecular pyruvate group, and R<NUM> is H or a monomolecular succinyl group.

In an embodiment not falling under the scope of protection, the heteropoly oligosaccharide of the present invention is prepared through enzymolysis of an exopolysaccharide Riclin under an action of β-glucanase, the exopolysaccharide Riclin being produced from Agrobacterium sp. ZCC3656 (CCTCC No.: M <NUM>). Details are given below:.

An exopolysaccharide Riclin produced from Agrobacterium sp. ZCC3656 (CCTCC No.: M <NUM>) is dissolved in an aqueous solution, β-glucanase is added to obtain a mixture. The mixture is placed in a thermostatic water bath at <NUM> for reaction and vibrated until the exopolysaccharide Riclin is enzymolyzed completely.

A solution obtained from the enzymolysis in step (<NUM>) is centrifuged to remove insoluble impurities and obtain a supernatant. A mixed solvent of chloroform and n-butanol at a volume ratio of <NUM>:<NUM> is added to the supernatant to remove proteins from the supernatant. The solution is vibrated vigorously and left to stand for stratification, the aqueous phase is taken and centrifuged to remove the protein layer, and the supernatant is retained. These steps are repeated several times until the protein layer is removed completely. Finally, <NUM>% ethanol is added to precipitate a heteropoly oligosaccharide.

The Agrobacterium sp. ZCC3656 has been disclosed in <CIT>.

In an embodiment of the present invention, the plants include but are not limited to crops and other plants, such as tobacco, wheat, tomato, potato, apple, strawberry, paddy, and soybean.

In an embodiment of the present invention, for the application of the heteropoly oligosaccharide in improving plant disease resistance, the plant disease resistance is the resistance to pathogenic bacteria infection in plants, including but not limited to the disease resistance to mosaic virus infection, disease resistance to Fusarium graminearum infection, disease resistance to Cladosporium fulvum infection, disease resistance to Phytophthora infestans infection, disease resistance to Marssonina mali infection and disease resistance to Pseudomonas solanacearum infection.

The present disclosure also provides a plant disease resistance inducer with an active ingredient containing the heteropoly oligosaccharide not falling under the scope of protection.

In the plant disease resistance inducer of the present disclosure, a concentration of the heteropoly oligosaccharide is <NUM>-<NUM>,<NUM>/L, and preferably <NUM>-<NUM>/L.

The heteropoly oligosaccharide of the present disclosure applied to crops and various other plants as a plant disease resistance inducer can significantly improve plant disease resistance, specifically as follows. <NUM>) When acting on tobacco leaves, it can obviously increase the concentration of hydrogen peroxide in tobacco leaves as the dose increases; <NUM>) When acting on lower epidermal cells of wheat leaves, it can obviously induce the release of hydrogen peroxide from cells of wheat leaves as the fluorescence intensity increases; <NUM>) When acting on tomato leaves, it can obviously improve activities of glucanase, chitinase and phenylalanine ammonia lyase continuously as the dose increases and the induction time prolongs; <NUM>) When acting on potato leaves infected with Phytophthora infestans patches, it can obviously reduce the damage of pathogenic bacteria to plants, and alleviate and even eliminate pathogenic bacteria infection.

Compared with the prior art, the present invention has the following advantages:.

The present invention will be further described below in detail in conjunction with the specific embodiments and accompanying drawings.

Agrobacterium sp. ZCC3656 used in the following embodiments has been disclosed in <CIT>.

Agrobacterium sp. ZCC3656 was inoculated into an Htm (containing <NUM> of sodium dihydrogen phosphate, <NUM> of anhydrous calcium chloride, <NUM> of magnesium chloride, <NUM> of ferrous sulfate, <NUM> of potassium nitrate, <NUM> of manganese sulfate, <NUM> of zinc chloride, <NUM>,<NUM> of water and <NUM> of sucrose, with a pH value of <NUM>-<NUM>) liquid medium, and cultured in a shaker at <NUM> and <NUM> rpm for <NUM>; a <NUM>-fold volume of industrial ethanol (<NUM>% ethanol) was added to the fermentation broth, and white filamentous polysaccharide precipitate was observed; the precipitate was collected through centrifugation at <NUM>,<NUM> × g and dried in a vacuum drying oven at <NUM> for <NUM>, and the solid polysaccharide was pulverized with a grinder to obtain a crude polysaccharide.

The polysaccharide was weighed and dissolved in an aqueous solution, β-glucanase was added, the mixture was uniformly mixed and allowed for reaction in a thermostatic water bath at <NUM>, and vibrated <NUM> later until the polysaccharide solution became not viscous any more.

The enzymolyzed solution was centrifuged at <NUM>,<NUM> rpm for <NUM> to remove insoluble impurities. Proteins were removed from the centrifuged supernatant by a Sevage method, i.e., by adding a <NUM>/<NUM> volume of a chloroform-n-butanol (<NUM>:<NUM>) mixture, the solution was vibrated vigorously for <NUM> and left to stand for stratification, the aqueous phase was centrifuged at <NUM>,<NUM> rpm for <NUM> to remove the protein layer, and the supernatant was transferred to a clean container. These operations were repeated several times until there was no protein layer. Finally, a <NUM>-fold volume of <NUM>% ethanol was added to precipitate a heteropoly oligosaccharide, and the heteropoly oligosaccharide precipitate was dried in the vacuum drying oven at <NUM>.

The test tobacco variety was Nicotiana X sanderae. The laboratory culture was conducted at a constant temperature of <NUM> for <NUM> of lightness and <NUM> of darkness, and the soil was kept moist. The test was divided into six groups: a control group sprayed with water, and groups sprayed with the heteropoly oligosaccharide at different concentrations (<NUM>/L, <NUM>/L, <NUM>/L, <NUM>/L and <NUM>/L, respectively). The leaves were collected within <NUM> after spraying to determine relevant signaling molecules.

<NUM>-<NUM> of fresh plant leaves were weighed and quickly ground into superfine powder in liquid nitrogen; <NUM> of acetone was added, the mixture was extracted under vortex vibration and centrifuged at <NUM>,<NUM> rpm for <NUM>, and the supernatant was transferred to a clean centrifuge tube; <NUM> of extract was taken and put in a <NUM> centrifuge tube, <NUM> of titanium tetrachloride reagent (<NUM> of titanium tetrachloride reagent contains <NUM> of concentrated hydrochloric acid, <NUM> of titanium tetrachloride and <NUM> of deionized water) was added, then <NUM> of concentrated ammonia water was added, the solution was centrifuged at <NUM>,<NUM> rpm for <NUM>, and the precipitate was collected; the precipitate was washed with acetone <NUM>-<NUM> times until the precipitate turned white; <NUM> of <NUM> H<NUM>SO<NUM> was added to the washed precipitate, and the precipitate was dissolved under vibration; <NUM>µL of sample was transferred to a <NUM>-well colorimetric plate, and the absorbance value at <NUM> was read. The results are shown in <FIG>.

The test tobacco variety was Nicotiana X sanderae. The outdoor culture was conducted, and the soil was kept moist. The test was divided into five groups: a control group sprayed with water, and groups sprayed with the heteropoly oligosaccharide at different concentrations (<NUM>/L, <NUM>/L, <NUM>/L and <NUM>/L, respectively). A tobacco mosaic virus was evenly sprayed on surfaces of tobacco leaves two days after the heteropoly oligosaccharide was sprayed, and the leaves were observed for infection. After the tobacco mosaic virus was sprayed, infection conditions of tobacco leaves were recorded every other week for three consecutive weeks. The results are shown in Table <NUM>.

To compare different groups, statistical analysis was conducted by an ANOVA statistical method. P<<NUM> was considered to indicate a significant difference.

It can be seen from <FIG> and Table <NUM> that the concentration of hydrogen peroxide in tobacco leaves obviously increased after the heteropoly oligosaccharide was sprayed. The concentration of hydrogen peroxide increased linearly and dependently as the concentration of the heteropoly oligosaccharide increased. According to the mosaic virus infection conditions of tobacco, the heteropoly oligosaccharide can obviously improve the resistance of tobacco to mosaic virus, enhance prevention and treatment effects on tobacco against the virus, and reduce the infection rate of tobacco leaves. Thus, the heteropoly oligosaccharide can be a potential biological pesticide for improving plant disease resistance.

The test wheat variety was Miannong No. <NUM>. The laboratory culture was conducted at a constant temperature of <NUM> for <NUM> of lightness and <NUM> of darkness, and the soil was kept moist.

The test was divided into three groups: a blank control group (i.e., using water as a control), a low-concentration heteropoly oligosaccharide group (with a final oligosaccharide concentration of <NUM>/L), and a high-concentration heteropoly oligosaccharide group (with a final oligosaccharide concentration of <NUM>/L). The fluorescence detection of H<NUM>O<NUM> was conducted by the following method: H<NUM>DCF-DA was prepared into a <NUM> mmol/L stock solution by DMSO, lower epidermis was carefully torn from plant leaves and incubated in a citric acid buffer with a pH value of <NUM> for <NUM>, then the citric acid buffer was replaced, H<NUM>DCF-DA was added until the final concentration reached <NUM>µmol/L, and incubation was conducted under horizontal shaking for <NUM>; upon completion of incubation, excess fluorescent dye was removed through <NUM> times of rinsing with the citric acid buffer; the lower epidermis of leaves was placed on a glass slide, and the fluorescence intensity was observed using a BP460-<NUM> laser color filter within a light wavelength range of <NUM>-<NUM>. The results are shown in <FIG>.

The test wheat variety was Miannong No. <NUM>. The outdoor culture was conducted, and the soil was kept moist. The test was divided into five groups: a control group sprayed with water, and groups sprayed with the heteropoly oligosaccharide at different concentrations (<NUM>/L, <NUM>/L, <NUM>/L and <NUM>/L, respectively). A Fusarium graminearum spore suspension was evenly sprayed on surfaces of wheat leaves two days after the heteropoly oligosaccharide was sprayed, and the leaves were observed for infection. Infection conditions of wheat leaves were recorded two weeks after the Fusarium graminearum spore suspension was sprayed. The results are shown in Table <NUM>. <MAT> <MAT> <MAT>.

It can be seen from <FIG> and Table <NUM> that the fluorescence intensity of hydrogen peroxide produced by stomatal cells was low when lower epidermal cells of wheat leaves were not induced by the heteropoly oligosaccharide, and the heteropoly oligosaccharide with a final concentration of <NUM>/L can obviously induce stomatal cells to produce a large amount of hydrogen peroxide. According to the scab infection conditions of wheat, spraying the heteropoly oligosaccharide can improve the resistance of wheat to Fusarium graminearum spores. After the heteropoly oligosaccharide was sprayed, the control effect significantly increased and the incidence obviously decreased. Thus, the heteropoly oligosaccharide can be a potential biological pesticide for improving plant disease resistance.

The test tomato variety was Huizhen No. <NUM>. The laboratory culture was conducted at a constant temperature of <NUM> for <NUM> of lightness and <NUM> of darkness, and the soil was kept moist.

The test was divided into three groups: a blank control group (i.e., using water as a control), a low-concentration heteropoly oligosaccharide group (with a final oligosaccharide concentration of <NUM>/L), and a high-concentration heteropoly oligosaccharide group (with a final oligosaccharide concentration of <NUM>/L). Samples were taken <NUM>, <NUM> and <NUM> after the heteropoly oligosaccharide was sprayed to determine activities of relevant proteases.

The test tomato variety was Huizhen No. <NUM>. The outdoor culture was conducted, and the soil was kept moist. The test was divided into five groups: a control group sprayed with water, and groups sprayed with the heteropoly oligosaccharide at different concentrations (<NUM>/L, <NUM>/L, <NUM>/L and <NUM>/L, respectively). A Cladosporium fulvum spore suspension was evenly sprayed on surfaces of tomato leaves two days after the heteropoly oligosaccharide was sprayed, and the leaves were observed for infection. Infection conditions of tomato leaves were recorded two weeks after the Cladosporium fulvum spore suspension was sprayed. The results are shown in Table <NUM>. <MAT> <MAT> <MAT>.

It can be seen from <FIG> and Table <NUM> that after the heteropoly oligosaccharide was sprayed onto tomato leaves, the activity of chitinase significantly increased at <NUM>, while activities of glucanase and phenylalanine ammonia lyase significantly increased at <NUM>. Spraying the heteropoly oligosaccharide to tomato can improve the resistance of tomato to Cladosporium fulvum spores. After the heteropoly oligosaccharide was sprayed, the control effect significantly increased and the incidence obviously decreased. Thus, the heteropoly oligosaccharide can be a potential biological pesticide for improving plant disease resistance.

The test potato variety was Fovorita. The laboratory culture was conducted at a constant temperature of <NUM> for <NUM> of lightness and <NUM> of darkness, and the soil was kept moist.

The test was divided into three groups: a blank control group (i.e., using water as a control), a low-concentration heteropoly oligosaccharide group (with a final oligosaccharide concentration of <NUM>/L), and a high-concentration heteropoly oligosaccharide group (with a final oligosaccharide concentration of <NUM>/L). Before the test, potato leaves were placed on a flat plate, and petioles were kept wet to prevent leaves from withering. The leaves were sprayed with different concentrations of the heteropoly oligosaccharide, kept wet and cultured at <NUM> for <NUM>. Phytophthora infestans patches were attached onto potato leaves. The leaves were kept away from light for <NUM>, and cultured for <NUM> of lightness and <NUM> of darkness. Two days later, infection conditions of leaves with patches were observed. The results are shown in <FIG>.

The test was divided into four groups: a blank control group (i.e., using water as a control), a low-concentration heteropoly oligosaccharide group (with a final oligosaccharide concentration of <NUM>/L), a medium-concentration heteropoly oligosaccharide group (with a final oligosaccharide concentration of <NUM>/L), and a high-concentration heteropoly oligosaccharide group (with a final oligosaccharide concentration of <NUM>/L). After a Phytophthora infestans spore suspension was sprayed, infection conditions of potato leaves were recorded every other week for three consecutive weeks.

It can be seen from <FIG> and Table <NUM> that after the heteropoly oligosaccharide was sprayed onto potato leaves, under the condition of infection with pathogenic bacteria patches, severe leaf infection was observed from the control group not treated with the heteropoly oligosaccharide, while leaves sprayed with the heteropoly oligosaccharide were slightly infected or even not infected. Spraying the heteropoly oligosaccharide to potato can improve the resistance of potato to Phytophthora infestans infection. After the heteropoly oligosaccharide was sprayed, the control effect significantly increased and the incidence significantly decreased. Thus, the oligosaccharide can favorably induce potato leaves to produce resistance and improve disease resistance, and can be a potential biological pesticide.

The test apple variety was Red Fuji. The outdoor culture was conducted, and the soil was kept moist. The test was performed during a lush growth of leaves and before apples were borne.

The test was divided into three groups: a blank control group (i.e., using water as a control), a low-concentration heteropoly oligosaccharide group (with a final oligosaccharide concentration of <NUM>/L), and a high-concentration heteropoly oligosaccharide group (with a final oligosaccharide concentration of <NUM>/L). A Marssonina mali spore suspension was evenly sprayed on surfaces of apple tree leaves two days after the heteropoly oligosaccharide was sprayed, and the leaves were observed for infection. After the Marssonina mali spore suspension was sprayed, infection conditions of apple tree leaves were recorded every other week for three consecutive weeks. The results are shown in Table <NUM>.

It can be seen from Table <NUM> that after the heteropoly oligosaccharide was sprayed onto apple tree leaves, under the condition of infection with the pathogenic bacteria spore suspension, severe leaf infection was observed from the control group not treated with the heteropoly oligosaccharide, while leaves sprayed with the heteropoly oligosaccharide were slightly or even not infected. Spraying the heteropoly oligosaccharide to apple trees can improve the resistance of apple trees to Marssonina mali. After the heteropoly oligosaccharide was sprayed, the incidence obviously decreased. Thus, the heteropoly oligosaccharide can be a potential biological pesticide for improving plant disease resistance.

The test strawberry variety was Ningxin. The outdoor culture was conducted, and the soil was kept moist. The test was performed during a lush growth of leaves and before strawberries were borne.

The test was divided into five groups: a control group sprayed with water, and groups sprayed with the heteropoly oligosaccharide at different concentrations (<NUM>/L, <NUM>/L, <NUM>/L and <NUM>/L, respectively). A strawberry Pseudomonas solanacearum suspension was evenly sprayed on surfaces of strawberry leaves two days after the heteropoly oligosaccharide was sprayed, and the leaves were observed for infection. Infection conditions of strawberry leaves were recorded two weeks after the Pseudomonas solanacearum suspension was sprayed. The results are shown in Table <NUM>. <MAT> <MAT> <MAT>.

Claim 1:
An application of a heteropoly oligosaccharide in improving disease resistance of a plant, wherein the heteropoly oligosaccharide comprises seven D-glucose residues and one D-galactose residue, characterized in that a structure of the heteropoly oligosaccharide molecule is represented as follows:
<CHM>
wherein R<NUM> is H or a monomolecular pyruvate group, and R<NUM> is H or a monomolecular succinyl group.