CN115474613A - Application of camellia saponin and/or ginkgo extract as bactericide for inhibiting rice nigrospora - Google Patents
Application of camellia saponin and/or ginkgo extract as bactericide for inhibiting rice nigrospora Download PDFInfo
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Abstract
The invention provides application of camellia saponin and/or a ginkgo extract as a bactericide for inhibiting rice nigrospora, and belongs to the technical field of bactericides. The single preparation of the camellia saponin and the ginkgo biloba extract has a remarkable inhibition effect on the nigrospora oryzae, so that both the saponin and the ginkgo biloba extract have the potential of preventing and treating plant diseases caused by the nigrospora oryzae. And the two are more advantageous in the aspect of environmental friendliness as plant extracts. The invention also screens the proportion of the camellia saponin and the ginkgo biloba extract with synergistic effect, improves the sterilization effect and provides a new medicament formula for the prevention and control of the nigrospora oryzae.
Description
Technical Field
The invention relates to the technical field of bactericides, and in particular relates to application of camellia saponin and/or a ginkgo extract as a bactericide for inhibiting Nigrospora oryzae.
Background
Nigrospora oryzae (Nigrospora oryzae) is a pathogenic fungus widely distributed in the global scope, can infect various plants such as corn, rice, wheat, sorghum, cotton, ginger, banyan and the like, and China is mainly distributed in main rice areas such as Heilongjiang, jiangsu, sichuan, hunan, guangdong and the like and can cause diseases such as rice ear rot, rice sheath blight, cotton black spot, dayflower leaf spot, rice branch blight, ginger leaf spot and the like.
Relatively few reports on the control of the bacterium exist at present. Plum-xueliqing et al report 75% oxime-tebuconazole water dispersible granule, 325 g.L -1 Benzyl azoxystrobin suspending agent, 250g L -1 The azoxystrobin suspension had a good plate-inhibiting effect on this bacterium (liturf, fufei, liturf bin, nigrospora oryzae biological characteristics and fungicide screening [ J]Changjiang river vegetables 2016, (6): 80-84.). The results of the test on the inhibitory effect of 10 bactericides on the rice black fungus showed that agents such as difenoconazole, fludioxonil, prochloraz manganese salt and benzyl propiconazole can be used as candidate agents for controlling Nigrospora oryzae (Feng Ericauli, cheng, zhu Xiaoyuan, etc.. 10 bactericides have indoor toxicity [ J ] to Nigrospora oryzae]Plant protection 2014,40 (4): 193-197.). In the aspect of biological control, sempere and the like prove that penicillium oxalicum can inhibit the growth of Neurospora oryzae, but the field has not been tested, verified, popularized and applied (SEMPERE F, SANTAMARINA M P. Suppression of Nigrospora oryzae (Berk).&Broome)Petch by An Aggressive Mycoparasite and Competitor,Penicillium oxalicum Currie&Thom[J]International Journal of Food Microbiology,2008,122 (1-2): 35-43). Therefore, it is necessary and urgent to develop new agents having preventive and therapeutic effects on nigrospora oryzae as soon as possible.
The sasanquasaponin is a natural surfactant with excellent performance and has multiple biological activities such as oxidation resistance and the like. In view of these activities, detergents, insecticides, various industrial emulsifiers, beer foaming agents, food industry emulsifiers and the like based on sasanquasaponin are currently being developed. In recent years, researches on the prevention and treatment of plant lethal pathogenic fungi by using sasanquasaponin are widely concerned, and 12 plant pathogenic bacteria are found by testing of sasanqua saponin and the like, wherein sasanqua saponin has a remarkable inhibition effect on hypha growth of 5 pathogenic bacteria of rice blast, penicillium citrinum of citrus, sclerotinia rot of tomato, phytophthora litchi, and corn small leaf spot, but has no inhibition effect on banana fusarium wilt, mango anthracnose, watermelon fusarium wilt, banana anthracnose and the like (sasanqua, chengxian, xuhanhong, and the like. The bacteriostatic activity of tea saponin on 12 plant pathogenic bacteria [ J ]. The university of agriculture news in Huazhong, 2013,32 (2): 50-53.). The inhibition effect of the sasanquasaponin on the rice nigrospora has not been reported at present.
Gingko is called plant activating stone and survives on the earth for hundreds of millions of years, and one of the important reasons is that the components contained in branches and leaves of the gingko have remarkable insect-resistant and bactericidal activity. It has been found that Ginkgo biloba leaves extract inhibits the infection of related hosts by Aspergillus niger, curvularia lunata, fusarium oxysporum, rhizoctonia solani (Liu XG, lu X, gao W, et al Structure, synthesis, biosynthesis, and activity of the structural compounds from Ginko bioloba L. Natural Product Reports,2022,39 (3): 474-511.) at low concentrations. However, the inhibiting effect of the ginkgo biloba extract on the Nigrospora oryzae is not reported at present.
Disclosure of Invention
The invention aims to provide a novel medicament formula for preventing and treating Nigrospora oryzae (Nigrospora oryzae), and provides a basis for screening a high-efficiency environment-friendly novel medicament for preventing and treating Nigrospora oryzae.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of camellia saponin and/or a ginkgo extract as a bactericide for inhibiting rice nigrospora.
Preferably, when the bactericide comprises camellia saponin alone, the bactericidal concentration of the camellia saponin is (6.3-10.5) × 10 -3 mg·L -1 。
Preferably, when the bactericide comprises the ginkgo extract alone, the bactericidal concentration of the ginkgo extract is (0.87-1.3) multiplied by 10 -3 mg·L -1 。
Preferably, when the bactericide comprises the camellia saponin and the ginkgo extract, the volume ratio of the camellia saponin to the ginkgo extract is 2.8-3.2: 3.8 to 7.2.
Preferably, the concentration of the sasanquasaponin is (6.3-10.5) multiplied by 10 -3 mg·L -1 The concentration of the ginkgo extract is (0.87-1.3) multiplied by 10 -3 mg·L -1 。
The single preparation of the camellia saponin and the ginkgo biloba extract has a remarkable inhibition effect on the nigrospora oryzae, so that both the saponin and the ginkgo biloba extract have the potential of preventing and treating plant diseases caused by the nigrospora oryzae. And both are more advantageous as plant extracts in terms of environmental friendliness. The invention also screens the proportion of the camellia saponin and the ginkgo biloba extract with synergistic effect, improves the sterilization effect and provides a new medicament formula for the prevention and control of the nigrospora oryzae.
Drawings
FIG. 1 shows the inhibitory effect of different concentrations of sasanquasaponin and ginkgo biloba extract on Nigrospora oryzae in example 1, wherein A-F are sasanquasaponin added to PDA medium at 9.45X 10 -3 mg·L -1 、8.40×10 -3 mg·L -1 、7.35×10 - 3 mg·L -1 、6.30×10 -3 mg·L -1 、5.25×10 -3 mg·L -1 、0mg·L -1 (ii) a G-L is prepared by adding semen Ginkgo extract into PDA culture medium to 1.30 × 10 -3 mg·L -1 、1.10×10 -3 mg·L -1 、0.90×10 -3 mg·L -1 、0.7×10 -3 mg·L -1 、0.50×10 -3 mg·L -1 、0mg·L -1 。
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
The following examples used Nigrospora oryzae (Nigrospora oryzae) collected from the leaves of diseased rice in the laboratory field of the science and technology institute of Hunan province, after conventional separation, purification and identification on PDA solid medium, the slant was stored at 4 ℃ for further use. Camellia saponin (hanqing biotechnology limited in Hunan), a ginkgo extract (CAS number: 90045-36-6, MDL number: MFCD00240717, cargo number: G832683-100G, specification: 10, purified from 1, and mainly comprises ginkgo total flavonoids and ginkgo terpene lactones), and a PDA culture medium (Hippocampus Biotechnology limited in Qingdao high-tech industrial garden).
The following examples the method for activating nigrospora oryzae was: inoculating the strain preserved by the slant solid into a PDA plate, culturing at the constant temperature of 28 ℃, and growing the bacterial colony to 2/3 of the culture dish.
Example 1
Determination of bacteriostatic activity of sasanquasaponin or ginkgo extract
(1) Preparing a camellia saponin concentration gradient culture medium: diluting the mother liquor of sasanquasaponin with water to 10.50 × 10 - 2 mg·L -1 、9.45×10 -2 mg·L -1 、8.40×10 -2 mg·L -1 、7.35×10 -2 mg·L -1 、6.30×10 -2 mg·L -1 、5.25×10 -2 mg·L -1 、4.20×10 -2 mg·L -1 The concentration of the solution was adjusted to 10.5X 10 by adding 1mL of the diluted solution and 9mL of PDA medium to the petri dish and mixing them -3 mg·L -1 、9.45×10 -3 mg·L -1 、8.40×10 -3 mg·L -1 、7.35×10 -3 mg·L -1 、6.30×10 -3 mg·L -1 、5.25×10 -3 mg·L -1 、4.20×10 -3 mg·L -1 PDA plates of sasanquasaponin at concentration, blank plus 1mL of sterile water and 9mL of medium, triplicate at each concentration, for a total of 24 plates.
(2) Preparing a ginkgo biloba extract concentration gradient culture medium: diluting semen Ginkgo extract with water to 1.30 × 10 - 2 mg·L -1 、1.10×10 -2 mg·L -1 、0.90×10 -2 mg·L -1 、0.70×10 -2 mg·L -1 、0.50×10 -2 mg·L -1 The concentration of the solution was 1mL of the diluted solution and 9mL of the PDA medium were added to the petri dish and mixed to prepare a mixture containing 1.30X 10 -3 mg·L -1 、1.10×10 -3 mg·L -1 、0.90×10 -3 mg·L -1 、0.70×10 -3 mg·L -1 、0.50×10 -3 mg·L -1 Concentrations of Ginkgo biloba extract PDA plates, blanks plus 1mL of sterile water and 9mL of medium, triplicate for each concentration, for a total of 18 plates.
(3) And (3) determining the antibacterial activity: the activated Nigrospora oryzae cake is punched by a 5mm puncher and inoculated to the center of a PDA (personal digital Assistant) plate containing the medicine, and the plate culture medium with the medicine and a blank plate is cultured at the constant temperature of 28 ℃. The observation was performed every 24 hours until the diameter of the blank colony reached 2/3 of the culture dish, and the colony diameter was measured once by cross-hatch method for each colony, and the average value was taken. And calculating the hypha growth inhibition rate according to the measured result. Then, a toxicity regression equation is drawn according to the concentration and the inhibition rate, and the effective intermediate concentration EC is calculated 50 。
Colony diameter (cm) = measured colony diameter-0.5;
hypha growth inhibition (%) = (blank colony diameter-treated colony diameter)/blank colony diameter × 100%.
The inhibition rate of the two bactericides on the hypha growth of the nigrospora oryzae is measured by a colony growth rate method, and the result is shown in table 1.
TABLE 1 inhibition rate of two fungicides on Nilapospora oryzae at different concentrations
Note: bacteriostatic rate = mean ± standard deviation (n = 3).
From table 1, it can be seen that both the sasanquasaponin and the ginkgo biloba extract have inhibitory effects on nigrospora oryzae, and the inhibitory effects are significant, and both exhibit concentration dependence. The gingko extract has the most obvious bacteriostatic effect on the rice melanosporum. The inhibitory effect of sasanquasaponin is increased by 1.0 × 10% and the concentration is increased by 10% each time -3 mg·L -1 Left and right; ginkgo extractThe inhibiting effect is increased by 0.20 × 10 for each 20% -3 mg·L -1 On the left and right, the side also shows that the ginkgo biloba extract has better bacteriostatic effect than the sasanquasaponin.
The growth conditions of the hyphae of the nigrospora oryzae also reflect the inhibition effect of the sasanquasaponin and the ginkgo extract (figure 1), the hyphae of the control groups (F and L) are plump and glittering, the internal structures are full and continuous, the whole growth state shows a healthy growth state, the hyphae of other groups (A-E and G-K) are obviously changed after being treated by the two bactericides, the hyphae are seriously wrinkled and shriveled, and the obvious fault phenomenon appears in the hyphae. The specific killing mechanism of the two on the Nigrospora oryzae is yet to be further researched.
The virulence determination results for a single agent against nigrospora oryzae are shown in table 2. EC of sasanquasaponin 50 Is 6.3X 10 -3 mg·L -1 EC of ginkgo biloba extract 50 Is 0.87X 10 -3 mg·L -1 EC for Nigrospora oryzae 50 Are all less than 0.1 mg.L -1 The toxicity effect is ideal. And both EC 50 Both at the 95% confidence limit, both chi-squared values were significant at a level greater than 0.150, and there were no heterogeneous factors in the confidence limits, indicating that the values were reliable. In terms of the magnitude of virulence, EC 50 The smaller the value, the more toxic it is, so the toxicity of the sasanquasaponin is less than that of the ginkgo extract, the latter is about 10 times of that of the former. Therefore, the effect of the ginkgo biloba extract on preventing and treating the rice nigrospora is far better than that of the sasanquasaponin.
TABLE 2 virulence determination of single agents against Nigrospora oryzae
Example 2
Screening of synergistic formulation of mixed medicament
Screening was performed by the Wadley method. Extracting with sasanquasaponin and semen GinkgoEC of taking out 50 Values (table 2) are as follows, in the following mixing ratios of 0. No drug was added as a blank control. After inoculation, the culture medium is placed at a constant temperature of 28 ℃. Observing every 24h until the diameter of the blank control colony reaches 2/3 of the diameter of the culture dish, measuring the diameter of the colony once by a cross method, taking the average value, and calculating the bacteriostasis rate. Calculating the Toxicity Ratio (TR) according to the predicted value and the actual value of the mixture ratio: TR = EC 50 Theoretical value/EC 50 The actual value is 100.
The interaction of the two is synergistic when TR is significantly >1, antagonistic when TR is significantly <1, and additive when TR = 1. The results are shown in tables 3 and 4.
TABLE 3 inhibitory Effect of the combination of Agents on Nigrospora oryzae
Note: bacteriostatic rate = mean ± standard deviation (n = 3).
As can be seen from the standard deviations in Table 3, the standard deviations of the single dose of sasanquasaponin, the single dose of ginkgo biloba extract and the mixed dose are mostly less than 0.1, which indicates that the stability of experimental data is good and the data result is reliable.
TABLE 4 synergistic effect of the drug mixture on Nigella oryzae
From the antifungal indices of table 4, divide by 3:7, the observed value is the highest and higher than the predicted value, the predicted values of the other mixture ratios are basically higher than the observed values, and the observed values have antagonism with different degrees and toxicity ratiosAre all made of<1. The actual inhibition ratio for the 3:7 ratio blend was 88.07% with a toxicity ratio of 2.84. Therefore, the optimal proportion of the mixed medicament is EC 50 Dosage of sasanquasaponin: ginkgo biloba extract = 3: 7.
The optimal proportion of sasanquasaponin determined according to the TR significant value is as follows: the ginkgo biloba extract = 3:7, and is diluted according to the proportion to obtain 1.956X 10 of 3 concentration gradients -3 、1.413×10 -3 、0.87×10 -3 mg·L -1 Mixing the reagents, preparing 12 PDA plates, culturing the bacterial cake, placing in a constant temperature incubator at 28 ℃ for 3 days, measuring the diameter of bacterial colony once, and taking the average value. Drawing a toxicity regression equation, solving the mixed agent toxicity regression equation Y = aX + b and r value, and solving EC 50 (A + B). The co-toxicity coefficient (CTC) was then determined. The results are shown in Table 5.
CTC＝[(EC 50 A/EC 50 (A+B)]/[(EC 50 A/EC 50 A)×Pa+(EC 50 A/EC 50 B)×Pb]×100。
In the formula, A and B respectively represent 2 medicament components of the sasanquasaponin and the ginkgo extract, and a and B are the content ratio of the sasanquasaponin and the ginkgo extract 2 in the mixed preparation. When CTC is >100, the interaction of the two has a synergistic effect; antagonism when CTC is more pronounced than 100; when CTC is approximately 100, the interactions are additive.
TABLE 5 determination of virulence of Agents Mixed to Nigrospora oryzae
As can be seen from Table 5, the mixture of sasanquasaponin and ginkgo biloba extract has synergistic inhibitory effect on Nigella sativa.
The single action of the sasanquasaponin and the ginkgo biloba extract and microorganisms are reported, but the inhibition of the rice nigrospora is not reported. The action mode and the action mechanism of the mixed sterilization of the sasanquasaponin and the ginkgo extract are still to be further researched.
From the above examples, it can be seen that the single dosage of sasanquasaponin and ginkgo biloba extract of the present invention has a significant inhibitory effect on nigrospora oryzae, which is equivalent to the effect of some commercial pesticides currently on the market, and therefore both of them have the potential of controlling plant diseases caused by nigrospora oryzae. And the two are more advantageous in the aspect of environmental friendliness as plant extracts. The invention also screens the proportion of the camellia saponin and the ginkgo biloba extract with synergistic effect, improves the sterilization effect and the environmental friendliness, and provides a new medicament formula for the prevention and control of the nigrospora oryzae.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (5)
1. Application of Camellia saponin and/or semen Ginkgo extract as bactericide for inhibiting Nigrospora oryzae is provided.
2. The use according to claim 1, wherein when the bactericide comprises sasanquasaponin alone, the sasanquasaponin has a bactericidal concentration of (6.3 to 10.5) × 10 -3 mg·L -1 。
3. The use of claim 1, wherein when the bactericidal agent comprises ginkgo extract alone, the bactericidal concentration of said ginkgo extract is (0.87-1.3) x 10 -3 mg·L -1 。
4. The use of claim 1, wherein when the bactericide comprises the sasanquasaponin and the ginkgo extract at the same time, the volume ratio of the sasanquasaponin to the ginkgo extract is 2.8-3.2: 3.8 to 7.2.
5. The use according to claim 4, wherein the concentration of sasanquasaponin is (6.3-10.5) x 10 - 3 mg·L -1 The concentration of the ginkgo extract is (0.87-1.3) multiplied by 10 -3 mg·L -1 。
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| 魏婷婷;崔晓芳;文旭;王丽;: "油茶粕中茶皂素纯化方法与抗菌活性研究", 中国油料作物学报, no. 06, pages 616 - 621 * |
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