CN110623005B - Compound plant extract for inhibiting bitter gourd fusarium wilt and preparation method thereof - Google Patents
Compound plant extract for inhibiting bitter gourd fusarium wilt and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a compound plant extract for inhibiting bitter gourd fusarium wilt, which is prepared by cleaning and sterilizing fresh garlic cloves, then crushing, then extracting with acetone, evaporating and concentrating to obtain garlic extract, and then dissolving the extract with deionized water to obtain garlic extract; drying stems and leaves of Chrysanthemum coronarium in the shade, pulverizing with a grinder to obtain Chrysanthemum coronarium powder, weighing the Chrysanthemum coronarium powder, extracting with ethanol solution for three times by ultrasonic wave, mixing the extractive solutions, evaporating to dryness to obtain paste, dissolving the extract with sterile distilled water, and dissolving the extract with deionized water to obtain Chrysanthemum coronarium extract; mixing the garlic extract and the garland chrysanthemum extract according to a certain proportion to obtain the garlic and garland chrysanthemum composite extract. The garlic and crowndaisy chrysanthemum composite extract has better prevention and treatment effect on bitter gourd fusarium wilt.
Description
Technical Field
The invention relates to a compound plant extract for inhibiting bitter gourd fusarium wilt and a preparation method thereof, in particular to a garlic extract, a crowndaisy chrysanthemum extract and application thereof in inhibiting bitter gourd fusarium wilt.
Background
In the production of the bitter gourds, the soil environment is worsened due to continuous cropping for years, continuous cropping obstacles become serious day by day, and soil-borne diseases are aggravated therewith. Bitter gourd Fusarium wilt is a typical soil-borne disease caused by Fusarium oxysporum bitter gourd specialized form (Fusarium oxysporum schl.f.sp.mordicae), and can cause bitter gourd to wither and wither in various bitter gourd planting areas in China, and finally, the bitter gourd dies. The common incidence rate of the bitter gourd fusarium wilt is reported to be 15% -25%, and the incidence rate is 60% -80% in serious cases, and a large number of dead vines appear in fields when the diseases are epidemic, so that the yield and the quality of the bitter gourd are seriously influenced. Aiming at the disease, the chemical agents mainly used in the production comprise iprodione, thiophanate-methyl, quintozene, carbendazim, hymexazol, chlorothalonil and the like. Because the balsam pear blight bacteria grow fast and have high suitability, the balsam pear blight bacteria can easily generate drug resistance to pesticides by continuously using the same chemical agent. And the chemical bactericides are difficult to degrade and easy to remain, and cause environmental pollution and food safety problems after long-term use. With the increasing attention of people to environmental protection consciousness, the plant source bactericide which is environment-friendly, nontoxic and harmless is pursued by the public.
The extraction and separation of bioactive substances from plants or plant products for controlling phytopathogens is one of the effective ways to develop and research novel pesticides. China is a big garlic producing country, the garlic yield, the garlic scale and the garlic planting area are the first in the world, the garlic is sufficient in raw material supply, and the garlic has good resource advantages in the aspect of developing plant source bactericides. The crowndaisy chrysanthemum has low requirement on the cultivation environment, can be cultivated in open field or in a greenhouse, and has short growth period. The plant bactericide has no pollution to the environment, is economic, efficient, nontoxic, reliable and environment-friendly, and is one of important directions for future research.
Therefore, the invention selects the garlic and the crowndaisy chrysanthemum with sufficient raw materials as the raw materials to prepare the economic, efficient, nontoxic and reliable plant bactericide which is applied to the prevention and treatment of the bitter gourd fusarium wilt.
Disclosure of Invention
The invention aims to provide a compound plant extract for inhibiting the fusarium wilt of bitter gourds, which is simple and convenient to operate, safe and time-saving, is extracted by natural plants, is green and environment-friendly, and has a lasting drug effect.
In order to achieve the purpose, the invention adopts the following technical scheme: a compound plant extract for inhibiting bitter gourd wilt is prepared by mixing Bulbus Allii extract and caulis et folium Chrysanthemi Segeti extract.
Preferably, the mass ratio of the garlic extract to the crowndaisy chrysanthemum extract is 2: 1-1: 1.
particularly preferably, the mass ratio of the garlic extract to the crowndaisy chrysanthemum extract is 2: 1.
specifically, the concentration of the crowndaisy chrysanthemum extract is 1 g/mL-1The concentration of the garlic extract is 1 g/mL-1。
A preparation method of a compound plant extract for inhibiting bitter gourd fusarium wilt comprises the following steps: cleaning fresh garlic cloves, sterilizing, crushing, extracting with acetone, evaporating and concentrating to obtain garlic extract, and dissolving the extract with deionized water to obtain garlic extract;
drying stems and leaves of Chrysanthemum coronarium in the shade, pulverizing with a grinder to obtain Chrysanthemum coronarium powder, weighing the Chrysanthemum coronarium powder, extracting with ethanol solution for three times by ultrasonic wave, mixing the extractive solutions, evaporating to dryness to obtain paste, dissolving the extract with sterile distilled water, and dissolving the extract with deionized water to obtain Chrysanthemum coronarium extract;
mixing the garlic extract and the garland chrysanthemum extract according to a certain proportion to obtain the garlic and garland chrysanthemum composite extract.
More specifically, the preparation steps are as follows:
soaking fresh Bulbus Allii in 75% ethanol for 2min, washing with sterile water for 3 times, drying surface water with absorbent paper, vacuum freeze drying, grinding into powder, pulverizing, and sieving with 40 mesh sieve;
weighing 500g Bulbus Allii powder, extracting with acetone under shaking for 3 times (20 hr each time) at a shaker speed of 140r min-1Combining the 3 times of extraction liquid, centrifuging, taking supernatant, and drying the filtrate by a freeze dryer. Concentrating by rotary evaporator to obtain Bulbus Allii extract; dissolving the extract with sterile distilled water, dissolving the extract with appropriate amount of deionized water, filtering with 0.22 μm filter membrane, and metering to 1 g/mL-1Storing in a refrigerator at 4 deg.C;
drying stems and leaves of caulis et folium Chrysanthemi Segeti in the shade, pulverizing with a grinder to obtain caulis et folium Chrysanthemi Segeti powder, weighing 5g of caulis et folium Chrysanthemi Segeti powder, adding 75% ethanol solution into a flask according to a material-to-liquid ratio of 1:10, extracting with ultrasonic wave at 45 deg.C for three times, mixing extractive solutions, rotary evaporating to dryness to obtain paste, dissolving the extract with sterile distilled water, dissolving the extract with appropriate amount of deionized water, filtering with 0.22 μm filter membrane, and fixing volume to 1 g.mL-1Storing in a refrigerator at 4 deg.C;
the concentration of the sample was measured to be 1 g/mL-1The crowndaisy chrysanthemum extract and the concentration of the crowndaisy chrysanthemum extract are 1 g/mL-1The garlic extract is prepared by mixing the components in a volume ratio of 1:1 to obtain 1 g/mL-1Garlic and crowndaisy chrysanthemum composite extractAnd (6) taking the object.
The invention adopts a growth rate method to determine the inhibition effect of the garlic extract, the crowndaisy chrysanthemum extract, the garlic and the crowndaisy chrysanthemum composite extract on the growth of the fusarium oxysporum bitter gourd specialized hyphae and the spore germination, and further carries out the seedling stage test of the garlic and the crowndaisy chrysanthemum extract for preventing and treating the bitter gourd fusarium wilt. Tests show that the garlic extract and the crowndaisy chrysanthemum extract both have an inhibiting effect on fusarium oxysporum, but the garlic extract and the crowndaisy chrysanthemum extract have a coordinated synergistic effect when mixed, the efficacy of the garlic and the crowndaisy chrysanthemum composite extract obtained by mixing the garlic extract and the crowndaisy chrysanthemum extract is superior to that of a single variety, the persistence of the efficacy is obviously improved, and the method is simple to operate, free of environmental pollution, economical, efficient, non-toxic and reliable, and has a good prevention and treatment effect on the bitter gourd fusarium wilt.
Drawings
FIG. 1 shows 0.1 g/mL-1Influence of the extract under the concentration on the growth of the specialized hyphae of the fusarium oxysporum balsam pear; in the figure, A: no extract was administered; b: compound extract of garlic and garland chrysanthemum; c: extract of caulis et folium Chrysanthemi Segeti; d: garlic extract.
FIG. 2 shows 0.1 g/mL-1Effect of the extract on the growth of fusarium oxysporum hyphae at concentrations.
FIG. 3 shows 0.05 g/mL-1Effect of the extract on the growth of fusarium oxysporum hyphae at concentrations.
FIG. 4 shows 0.025 g/mL-1Effect of the extract on the growth of fusarium oxysporum hyphae at concentrations.
FIG. 5 shows 0.1 g/mL-1Effect of the extract on the germination of fusarium oxysporum spores at concentrations.
FIG. 6 shows 0.05 g/mL-1Effect of the extract on the germination of fusarium oxysporum spores at concentrations.
FIG. 7 shows 0.025 g/mL-1Effect of the extract on the germination of fusarium oxysporum spores at concentrations.
FIG. 8 shows the results of field experiments on the effect of garlic and crowndaisy chrysanthemum composite extract on bitter gourd seedlings.
FIG. 9 shows the effect of garlic and crowndaisy chrysanthemum composite extract on the leaves of bitter gourd seedlings.
In fig. 8 and 9: a, applying a garlic and crowndaisy chrysanthemum composite extract B: no extract was applied.
FIG. 10 is a microscopic photograph of the diseased leaves of balsam pear seedlings without the application of the extract.
FIG. 11 is a microscope photograph of young balsam pear seedlings without disease after being applied with the garland chrysanthemum morifolium composite extract.
FIG. 12 shows the results of the test material inoculation identification.
Detailed Description
The present invention will be described in further detail with reference to examples.
Soaking fresh Bulbus Allii in 75% ethanol for 2min, washing with sterile water for 3 times, drying surface water with absorbent paper, vacuum freeze drying, grinding into powder, pulverizing, and sieving with 40 mesh sieve;
weighing 500g Bulbus Allii powder, extracting with acetone under shaking for 3 times (20 hr each time) at a shaker speed of 140r min-1Combining the 3 times of extraction liquid, centrifuging, taking supernatant, and drying the filtrate by a freeze dryer. Concentrating by rotary evaporator to obtain Bulbus Allii extract. Dissolving the extract with sterile distilled water, dissolving the extract with appropriate amount of deionized water, filtering with 0.22 μm filter membrane, and metering to 1 g/mL-1Storing in refrigerator at 4 deg.C to obtain Bulbus Allii extract.
Drying stems and leaves of caulis et folium Chrysanthemi Segeti in the shade, pulverizing with a grinder to obtain caulis et folium Chrysanthemi Segeti powder, weighing 5g of caulis et folium Chrysanthemi Segeti powder, adding 75% ethanol solution into a flask according to a material-to-liquid ratio of 1:10, extracting with ultrasonic wave at 45 deg.C for three times, mixing extractive solutions, rotary evaporating to dryness to obtain paste, dissolving the extract with sterile distilled water, dissolving the extract with appropriate amount of deionized water, filtering with 0.22 μm filter membrane, and fixing volume to 1 g.mL-1Storing in refrigerator at 4 deg.C to obtain caulis et folium Chrysanthemi Segeti extract.
The concentration of the sample was measured to be 1 g/mL-1The crowndaisy chrysanthemum extract and the concentration of the crowndaisy chrysanthemum extract are 1 g/mL-1The garlic extract is prepared from the following components in a mass ratio of 1:1 to obtain 1 g/mL-1The compound extract of garlic and garland chrysanthemum.
1. Activity measurement of garlic and garland chrysanthemum extract on fusarium oxysporum hypha growth
Medicated bagPreparing a nutrient medium: placing the extract sterilized by filtration with filter membrane into sterilized test tube, adding PDA culture medium, shaking, and placing into sterilized culture dish to obtain the final product with concentration of 0.1, 0.05, 0.025 g.mL-1Solid culture medium with medicine.
The growth rate method is adopted to measure the inhibition effect of the extract on the growth of pathogenic bacteria hyphae. Cutting 5mm of fungus cake with a puncher, placing on the culture medium with medicine, taking PDA culture medium containing distilled water as control, placing in a constant temperature incubator at 28 deg.C for culture, measuring colony diameter by cross method after 96 hr, taking average value, repeating for 3 times, and calculating hypha growth inhibition rate.
As can be seen from fig. 1, compared with sample a without the extract, the garlic extract, the garland chrysanthemum extract, and the garlic and garland chrysanthemum composite extract all have stronger allelopathy inhibition effect on the growth of fusarium oxysporum within the test treatment concentration range, and the garlic extract (sample D) has stronger inhibition effect on fusarium oxysporum than the garland chrysanthemum extract (sample C) under the same concentration. However, the compound extract of garlic and chrysanthemum coronarium (sample B) has stronger inhibition effect on fusarium oxysporum than the compound extract of garlic and chrysanthemum coronarium, which indicates that the compound extract of garlic and chrysanthemum coronarium has coordination and synergism effect, so that the compound extract of garlic and chrysanthemum coronarium has stronger inhibition effect on fusarium oxysporum under the same concentration.
Allelopathy effect index (RI) is used to represent allelopathy effect of each extract treatment on hypha growth. The results are shown in FIGS. 2-4, where C is the control value, T is the treatment value, and RI is the allelochemical effect, and RI is 1-C/T when T ≧ C, and T ≦ C, RI ═ T/C-1. RI > 0 is promoting effect, and RI < 0 is inhibiting effect. The extract content of caulis et folium Chrysanthemi Segeti is 0.25 g/mL-1The garlic extract has a chemosensory promoting effect at the treatment concentration, but shows an inhibiting effect with the increase of the treatment concentration, and relatively, the garlic extract has a stronger inhibiting effect on fusarium oxysporum. The compound extract of garlic and chrysanthemum coronarium is more than that of garlic and chrysanthemum coronariumThe extract has stronger fusarium oxysporum inhibition effect, and the longer the time is, the more obvious the difference is, which indicates that the mixture of the garlic extract and the garland chrysanthemum extract is beneficial to improving the durability of the fusarium oxysporum inhibition effect.
2. Effect of extracts on Fusarium oxysporum spore germination
The spore concentration is about 1.0 × 103cfu·mL-1A suspension of (a). Mixing Bulbus Allii extract, caulis et folium Chrysanthemi Segeti extract and Bulbus Allii, caulis et folium Chrysanthemi Segeti composite extract 1 g/mL-1Diluting to 0.1, 0.05, 0.025 g/mL-1The spore suspension was pipetted into a concave slide with different concentrations of root exudate solution. Each treatment was set to 5 replicates. Carrying out moisture-preserving culture at 25 ℃, observing the spore germination condition, wherein the calculation formula of the spore germination rate is as follows:
spore germination rate (%) ═ number of germinated spores/total number of spores × 100
As shown in figures 5-7, the garlic extract, the crowndaisy chrysanthemum extract and the garlic and crowndaisy chrysanthemum composite extract have different allelopathic effects on the germination of fusarium oxysporum spores and show stronger inhibition effects, the garlic extract has stronger inhibition effect than the crowndaisy chrysanthemum extract, and the garlic and crowndaisy chrysanthemum composite extract has stronger inhibition effect than the garlic extract. The test result shows that under the test treatment concentration, the higher the treatment concentration is, the stronger the allelopathy inhibiting effect is, the lower the germination rate of fusarium oxysporum spores is, and the allelopathy effect and the treatment time are inversely related.
3. Field test of garlic crowndaisy chrysanthemum composite extract after artificial inoculation
Inoculating the vigorous blight hyphae into a liquid culture medium, and performing shake culture at 28 ℃ for 3-5 days. Filtering the culture solution with double-layer gauze to remove mycelium, centrifuging the filtrate, and removing the supernatant. Calculating spore concentration by hemocytometer, and adjusting to 106cfu·mL-1The concentration of the bacterial liquid is used for inoculation identification. And (3) carrying out artificial inoculation identification on bitter gourd seedlings by adopting a root soaking transplanting method when the three leaves are in one heart. As shown in fig. 8 and 9, 12d after inoculation, seedlings a grew normally, seedlings B yellow leaves, leaf veins, and some leaf wilting, which are typical symptoms of blight. FIG. 10 shows the young balsam pear without the extractMicroscope observation photo after seedling leaf outbreak, fig. 11 is microscope observation photo of balsam pear seedling without outbreak of disease applied with garlic crowndaisy chrysanthemum composite extract. The result shows that the garlic crowndaisy chrysanthemum composite extract has good bitter gourd fusarium wilt prevention and treatment effect.
4. Test of drug effect of garlic crowndaisy chrysanthemum composite extract with different proportions
The test material is a high-sensitivity variety LK0901 and a high-resistance variety BK0604, the Fusarium oxysporum to be tested is collected from an experimental base of vegetable and flower research institute of agricultural academy of agricultural sciences in Jiangxi, and the test material is inoculated and identified as a Fusarium oxysporum bitter gourd specialized type (Fusarium oxysporum Schl.f.sp.mordicae) as shown in FIG. 12.
Culturing bitter gourd seedlings: soaking the seeds of the balsam pear for 17 hours, wrapping the seeds by gauze, putting the wrapped seeds in a constant-temperature incubator at 32 ℃ for accelerating germination, and sowing the seeds in a 32-hole plug tray after the seeds are exposed to the white. Each line was replicated 3 times, 20 strains each time.
Inoculating and irrigating roots: pulling out when seedling of fructus Momordicae Charantiae grows to two leaves and one heart, cleaning root matrix, and adding spore suspension (1 × 10) of Fusarium oxysporum6cfu·mL-1) Soaking for 30min, transplanting sterile matrix seed, applying 100mL of compound extract of Bulbus Allii and caulis et folium Chrysanthemi Segeti at different ratio to the matrix, and placing in artificial climate box (temperature 28 deg.C, illumination for 12 h.d)-1Relative humidity 70%). The garlic crowndaisy chrysanthemum composite extract is applied once every 3 days for four times. The disease condition of the plants is classified into 5 grades. Grade 0 of the disease condition: no symptoms; stage I: 1-2 leaves of the Chinese magnoliavine obviously turn yellow; II stage: the cotyledon becomes yellow and wilted, and the vascular bundle slightly changes color; grade III: the cotyledon withers, the true leaf turns yellow, and the vascular bundle is brown; stage IV: the whole plant withers or withers, and the vascular bundle is brown. The disease-resistant plants are on the 0 and I grades, and the disease-sensitive plants are on the II-IV grades. The disease resistance of the variety is graded according to Disease Index (DI), the disease resistance of the variety is graded as 5, the disease index is 0-15 High Resistance (HR), 15-30 resistance (R), 30-50 Medium Resistance (MR), 50-70 susceptibility (S) and 70-100 High Susceptibility (HS).
Disease index ∑ (number of diseased plants × representative value of the grade)/(total number of plants × representative of highest grade) × 100.
Wherein, the representative value of the level is the disease level of the diseased plants, and the highest level is 4 levels.
TABLE 1 Garlic crowndaisy chrysanthemum composite extract ratio
TABLE 2 disease indices in different proportions
As can be seen from the above table, in both the high-susceptibility variety LK0901 and the high-resistance variety BK0604, the mass ratio of the garlic extract to the crowndaisy chrysanthemum extract is 2: 1-1: 1 hour, the garlic crowndaisy chrysanthemum composite extract has a good effect of inhibiting the wilt disease, and particularly, the mass ratio of the garlic extract to the crowndaisy chrysanthemum extract is 2: the effect of 1 is particularly prominent.
The invention adopts a growth rate method to determine the inhibition effect of the garlic extract, the crowndaisy chrysanthemum extract, the garlic and the crowndaisy chrysanthemum composite extract on the growth of the fusarium oxysporum bitter gourd specialized hyphae and the spore germination, and further carries out the seedling stage test of the garlic and the crowndaisy chrysanthemum extract for preventing and treating the bitter gourd fusarium wilt. The test shows that the garlic and garland chrysanthemum composite extract has good effect of preventing and treating bitter gourd fusarium wilt.
The above embodiment data shows the embodiments of the present invention, but the present invention is not limited to the above embodiment data, and simple parameter adjustment based on the above embodiment data is also within the scope of the present invention.
Claims (1)
1. A compound plant extract for inhibiting bitter gourd fusarium wilt is characterized in that: the garlic extract and the crowndaisy chrysanthemum extract are mixed, and the mass ratio of the garlic extract to the crowndaisy chrysanthemum extract is 1:1, the concentration of the crowndaisy chrysanthemum extract is 1 g.mL-1The concentration of the garlic extract is 1 g/mL-1；
The preparation process comprises the following steps:
soaking fresh Bulbus Allii in 75% ethanol for 2min, washing with sterile water for 3 times, drying surface water with absorbent paper, vacuum freeze drying, grinding into powder, pulverizing, and sieving with 40 mesh sieve;
weighing 500g of garlic powder, extracting with acetone under shaking for 3 times, each time for 20h, setting the speed of a shaking table at 140 r/min, mixing the 3 times of extractive solutions, centrifuging, collecting supernatant, and drying the filtrate with a freeze dryer; concentrating by rotary evaporator to obtain Bulbus Allii extract; dissolving the extract with sterile distilled water, dissolving the extract with an appropriate amount of deionized water, filtering with a 0.22 mu m filter membrane, and metering to 1 g/mL-1Storing in a refrigerator at 4 deg.C;
drying stems and leaves of crowndaisy chrysanthemum in the shade, crushing the stems and leaves of the crowndaisy chrysanthemum by using a grinder to obtain crowndaisy chrysanthemum powder, weighing 5g of the crowndaisy chrysanthemum powder into a triangular flask, adding 75% ethanol solution into the flask according to the material-liquid ratio of 1:10, performing ultrasonic extraction for three times at 45 ℃, combining extract liquor, performing rotary evaporation and evaporation to dryness to obtain paste, dissolving the extract by using sterile distilled water, dissolving the extract by using a proper amount of deionized water, filtering the extract by using a 0.22 mu m filter membrane, and fixing the volume to 1 g.mL-1Storing in a refrigerator at 4 deg.C;
the concentration of the sample was measured to be 1 g/mL-1The crowndaisy chrysanthemum extract and the concentration of the crowndaisy chrysanthemum extract are 1 g/mL-1The garlic extract is prepared by mixing the components in a volume ratio of 1:1 to obtain 1 g/mL-1The compound extract of garlic and garland chrysanthemum.
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