CN100431423C - Application of Merremia boisiana extract in preparing bactericide - Google Patents
Application of Merremia boisiana extract in preparing bactericide Download PDFInfo
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Abstract
The present invention provides an application of a Merremia boisiana extract in preparing bactericides. The Merremia boisiana extract has broad-spectrum high-efficiency antibacterial activity; the Merremia boisiana extract has bacteriostatic action on plant pathogenic fungi, food mycete and plant pathogenic bacteria, and has very high control efficiency on rice sheath blight diseases and cucumber downy mildew in major plant diseases. The extract has the advantages of safe to crops, easy biodegradation, no residue and no pollution because the extract is a natural substance. Simultaneously, the mechanism of action on plant pathogenic bacterium is different from various commonly-used bactericides, and therefore, the Merremia boisiana extract is helpful to solve the problem of serious bactericide resistance of bactericides in actual production. Therefore, the present invention not only provides a novel good bactericide for production, and also opens up a new path of controlling and treating the serious spread of noxious plants, Merremia boisiana, in the forest ecological environment.
Description
Technical field
The invention belongs to pesticide field, be specifically related to the application of Merremia boisiana extract in the preparation bactericide, relate to the preparation method of golden clock rattan plant extracts simultaneously.
Background technology
As everyone knows, agricultural chemicals is one of guarantee means of control of plant disease, even in today, though the utilization of disease-resistant gene has obtained development fast under the molecular biology relevant technologies promotes, but chemical pesticide still plays in control of plant disease can not substituted role.1934 the bactericide tmtd (Thiram) appearance, the epoch of having opened the organic synthesis bactericide.From then on, the control of plant disease is more efficient, and the bactericide of chemosynthesis has been made positive contribution in control of plant disease.But since the sixties in last century, the public more and more payes attention to the pollution problem of environment toxicity of pesticide and agricultural chemicals.The bactericide kind of some toxicity height, residual length, disabled in succession as organic mercurial and difoltan (captafol) etc.To the nineties in last century, people are more and more higher to food security and requirement on environmental protection.1996; the environmental protection bill that Americanologist is crossed, with 4 kinds of important protective fungicides: maneb (maneb), mancozeb (mancozeb), tpn (chlorothalonil) and captan (captan) are classified the B2 carcinogen as and are forbidden using in the U.S..Subsequently, corresponding forbidding restriction has also been made by European Union.Now, particularly after China had added WTO, the residue of pesticide problem had become the international trade barrier that the restriction agricultural products in China exports; Some developed country even clearly forbid once using the agricultural product of certain Synthetic Organic Chemistry pesticide species from China's import.So in the face of the impact of economic globalization that enlarges day by day, seeking efficient, safety and environment amenable agricultural chemicals (comprising bactericide) is the vast pesticide science worker's of China opportunities and challenges.
Since nineteen sixty for last systemics commercialization since, the drug-fast problem of bactericide has become the serious problems in the production, the bactericide kind that some are important all is just to occur serious pesticide resistance problem after using 1-2 on producing such as carbendazim (carbendazim) and metalaxyl (metalaxyl) etc.According to the relevant research report of units such as Agricultural University Of Nanjing, various sterilization agent such as carbendazim serious pesticide resistance problem occurred in China.For the drug-fast improvement of bactericide, main measure comprises: (1) is mixed with traditional protectant and is used or be used alternatingly; (2) use the different bactericide of the mechanism of action.Because problem above-mentioned, present most important traditional protection agent is all forbidden by developed countries such as America and Europes.Therefore, use the bactericide of the different mechanisms of action just to become one of major measure that solves bactericide pesticide resistance problem.But at present in the world, the bactericide kind that the mechanism of action that can obtain is different is also fewer.Therefore, find that the new bactericide with different mechanisms of action is another opportunities and challenges of bactericide scientific worker.
The compound of vegetalitas or plant resource just is applied on the human medicine and agricultural chemicals since ancient times widely.In developing country, plant is the main source of medicine, has 80% population mainly to depend on plant and the plant extract life and health that becomes to assign to safeguard in the world, even in the U.S., also there is prescription medicine important more than 120 kinds to derive from plant, accounts for 25% of the total consumption of united states drug greatly.Most important three major types insecticide: pyrethroid, carbamates and the anabasine that develops rapidly in recent years, its synthetic lead compound is all from higher plant; And rotenone, different goat's horn are turned round all commercializations of many new vegetable insecticide such as glucoside and nimbin.1998, Meng Zhaoli etc. isolate from ginkgo had very highly active compound to phytopathogen, developed " green Supreme Being " agricultural bactericidal agent series products.Because advantages such as botanical pesticide from natural, has easy biological degradation, and is free from environmental pollution.At present, from plant, seek antimicrobial compound and develop the attention that the vegetalitas bactericide just more and more is subjected to countries in the world.
Gold clock rattan (Merremia boisiana (Gagnep.) van Ooststr.) is Convolvulaceae (Convolvulaceae), jasmine goldenrain tree Calamus (the yellow grass of fish belongs to) (Merremia Dennst.Ex Endl.) plant, and Chinese name also claims to spend more mountain pig dish or false sweet potato.Gold clock rattan is distributed in Indonesian Kalimantan, and Malay sand is got over always, ground such as North Vietnam (former Tokyo, Sabah, peace earlier) and Laos the north; China is distributed in Hainan, Guangxi, Yunnan and Guangdong.Gold clock rattan is perennial, feminine gender, the bejuco that happiness is cloudy.In recent years, golden clock rattan presents sudden large tracts of land harm in the Guangzhou, Guangdong, be the forestry noxious plant, is ecological destructive species.The fertility of gold clock rattan is strong, fast growth, and multi-branched is intertwinded forest, covers tree crown, causes forest withered in flakes, and forest ecology is destroyed greatly.Reported first in 1994 is the forest farm in suburbs, Guangzhou, Guangdong harm forest, and harm is serious day by day in recent years.To the end of the year 2004,300 hectares of mountain forests are injured on mountain, screen-like mountain peak, Tianhe District Taihe county, Guangzhou.Thereby, Guangzhou and become the new distribution center that emerges of golden clock rattan in the neighbourhood just gradually.For the harm of Jin Zhongteng, control is difficulty very.Manually prevent and kill off all undesirable with the method effect of using weed killer herbicide: the rattan that breaks apart by chopping grows adventive root and rudiment bar very soon as long as contact with ground; Use sulfometuron-methyl and multiple weedicide such as the rattan prestige of going out are sprayed, only can the tender cauline leaf of fragmentation unit children, other rhizome all there is not obvious effect.Therefore, how Jin Zhongteng being carried out comprehensive utilization and be used for controlling golden clock rattan outburst and spread, seek the effective new method that control solves the harm of golden clock rattan, thereby turn bane into boon, is the problem of demanding urgently researching and solving in the present production of forestry.
Summary of the invention
(1) technical problem that will solve
The object of the present invention is to provide the application of Merremia boisiana extract in the preparation bactericide, the preparation method of Merremia boisiana extract is provided simultaneously.
(2) technical scheme
The present patent application people is by suppressing the bacteriostasis of various plants pathogen and the further investigation of bacteriostasis mechanism to Merremia boisiana extract, find that its extract not only all has stripped bacteriostatic activity very high and very wide spectrum to the various plants pathogen, and multiple important plant disease is had good live body protective effect; And, gold clock rattan plant extracts is different with the mechanism of action of more existing important bactericide kinds (as metalaxyl, dimethomorph, Fluoxastrobin etc.) to the mechanism of action of phytopathogen, helps to administer the in fact serious pesticide resistance problem of bactericide of producing.
On the basis of above-mentioned research, the invention provides the application of Merremia boisiana extract in the preparation bactericide.
Wherein Merremia boisiana extract is the extract of root, stem and/or the blade of golden clock rattan plant.Used extraction solvent is water or middle polarity to high polarity organic solvent such as chloroform, ethyl acetate, acetone, methyl alcohol, ethanol, n-butanol etc.
Described Merremia boisiana extract is by solvent extraction method, steam distillation or sublimed method preparation.Wherein solvent extraction method comprises: the dipping bubble extracts under the room temperature, percolation extracts, decocting method extracts; Can also adopt some new extracting method, as methods such as supercritical fluid extraction extraction, Microwave Extraction, Extraction by Ultrasound, Enzymatic Extraction, half bionic extraction, broken extraction or airblasting extractions.
Application of the present invention can be prepared into Merremia boisiana extract aqua (aqueoussolutions), soluble powder (soluble powders), wetting powder (wettablepowders), suspension concentrates (suspensions concentrates) and granula (granules).
More than the preparation method of various formulations be the conventional method of this area, the adjuvant used usual auxiliaries that is in the pesticide field in each formulation preparation process.The concentration of the GOLD FROM PLATING SOLUTION clock boisiana extract that each formulation is prepared in use is 1 μ g/ml-1000mg/ml.
Through experimental results show that: Merremia boisiana extract all has very strong stripped antibacterial activity to various plants pathogen such as lower fungus, higher fungi and bacterium, shows wider fungicidal spectrum; Gold clock rattan methanolic extract has good live body prophylaxis effect adding after a little auxiliary (as polysorbas20 etc.) makes aqua to cucumber downy mildew, and protection effect is suitable with the commercially available agricultural chemical metalaxyl-mn-zn, and plant is not had poisoning.Gold clock rattan n-butanol extract is adding after a little auxiliary (as polysorbas20 etc.) makes aqua, rice sheath blight disease had good prophylaxis effect, its effect and commercially available agricultural chemical Fluoxastrobin (azoxystrobin, the commodity of Syngenta Co.,Ltd are that 25% Amici reaches suspending agent), the control efficiency of carbendazim is suitable, and plant do not had poisoning.The bacteriostasis mode and the mechanism of the ethyl acetate extract of gold clock rattan: cause cucumber phytophthora root rot mycelia deformity: branch increases, tubbiness and expanding; The peronophythora litchi bacterium is had bactericidal action, show as the sprouting that suppresses spore, and the useful effect concentration of the spore germination that suppresses to stop is low to moderate 50 μ g/ml; Especially, can make the zoospore explosion.
Bacteriostatic active ingredients can also further adopt phytochemical method to separate and identify in the gold clock rattan, to find new compound with sterilization and antibacterial activity, and can be on this basis, with this compound structure is template, carry out molecular structure alteration, study its structure-activity relationship,, and it is developed to new bactericide kind so that obtain more highly active compound.
(3) beneficial effect
Of the present invention is that the new broad-spectrum high efficacy bactericide of active ingredient has following characteristics with the Merremia boisiana extract:
(1) extracting method is simple;
(2) active ingredient is the natural antibacterial compound, and is nontoxic, have no side effect, free from environmental pollution;
(3) have broad spectrum of activity, the bactericidal activity height, usage amount is low, and protection effect is remarkable;
(4) bactericidal action mechanism uniqueness helps to administer the in fact serious pesticide resistance problem of bactericide of producing;
(5) raw material sources are wide, and turn bane into boon.
Embodiment
Can further be expressly understood the present invention by specific embodiments of the invention given below, but following embodiment not a limitation of the invention.
Embodiment 1
Golden clock rattan plant is gathered respectively according to old rattan (containing cortex) and green tender rattan (containing blade), dry in the shade (or oven dry under 50 degree Celsius), mechanical crushing, the 40-60 order sieves.Crushed material at ambient temperature, organic solvent methyl alcohol cold soaking with 5 times of amounts extracts 3 times, soak time is respectively 3 days, 2 days and 1 day, filter successively and collect filtrate, merge 3 times filtrate, be lower than concentrating under reduced pressure under 55 degree, remove methyl alcohol in Celsius temperature, can obtain golden clock rattan methyl alcohol crude extract dry matter, stored refrigerated, standby.
Embodiment 2
The methyl alcohol crude extract of the old rattan of golden clock rattan (containing cortex) that experimental example 1 is obtained is with 80% dissolve with ethanol of 5 times of amounts, gained solution is put in the separatory funnel, at ambient temperature, adding extracts with the organic solvent benzinum of volume, emit the ethanolic solution of lower floor earlier, emit the petroleum ether solution on upper strata again.Petroleum ether solution is lower than concentrating under reduced pressure under 55 degree in Celsius temperature, reclaim benzinum after, will join with the benzinum of volume once more and extract in the ethanolic solution and the concentrated recovery of benzinum, till the color of petroleum ether extraction liquid becomes water white transparency.Petroleum ether solution promptly obtains ligroin extraction, stored refrigerated, standby after concentrating.
Remaining ethanolic solution is earlier under the temperature of 55 degree Celsius, and concentrating under reduced pressure boils off and adds entry behind the ethanol that is contained and be supplemented to original volume.The aqueous solution is put in the separatory funnel, and the organic solvent chloroform that adds with volume extracts.Concentrate and reclaim chloroform and then extraction, concrete grammar and condition are the same, till the color of chloroform extraction liquid becomes water white transparency.Chloroform extraction liquid promptly obtains the chloroform extract of golden clock rattan after concentrating under reduced pressure is removed chloroform.
According to said method and condition, use organic solvent ethyl acetate and n-butanol to extract respectively successively with volume, concentrate and reclaim and extraction again.Just can obtain ethyl acetate extract and the n-butanol extract of golden clock rattan respectively.The Sheng Xia aqueous solution at last, natural air drying is perhaps dried under the temperature of 55 degree Celsius, can obtain golden clock rattan water extract.
Embodiment 3
Take by weighing golden clock rattan methyl alcohol crude extract 1 gram of embodiment 1, be dissolved in water and be settled to 100 milliliters, and add 100 μ l polysorbas20s, can be prepared into 1% gold medal clock rattan methyl alcohol crude extract aqua.In like manner, take by weighing golden clock rattan n-butanol extract 1 gram of embodiment 2, be dissolved in water and be settled to 100 milliliters, and add 100 μ l polysorbas20s, can be prepared into 1% gold medal clock rattan n-butanol extract aqua.
The methyl alcohol crude extract of embodiment 4 gold medal clock rattan plant different parts is to the bacteriostatic activity of plant pathogenic fungi mycelial growth
1. for the examination pathogen
Rice blast fungus (Magnaporthe grisea), peronophythora litchi bacterium (Peronophthora litchii), rhizoctonia (Rhizoctonia solani), cucumber phytophthora (Phytophthora melonis), Glorosprium musarum Cookeet Mass (Colletotrichum musae), withered germ of water-melon (Fusarium oxysporium f.sp.vineum), tobacco leaf point mould (Phyllostictanicotinanae), pineapple black rot (Ceratocystis fimbriata), tomato early blight bacterium (Alternaria solani), pythium spp (Pythium spp.), Phomopsis citri bacterium (Phomopsiscitri), Luo Shi southern blight bacterium (Sclerotium rolfsii).
2. test method
Adopt the growth rate method of toxic medium to measure the inhibitory action of golden clock rattan plant methyl alcohol crude extract to the phytopathogen mycelial growth.Concrete grammar see reference document (Huang Guirong, Li Youzhi, Xu Dagao, Pan Ruqian, Xu Hanhong.9 kinds of western Hunan plant methanolic extract antifungal activities [J].Zhong Kai agrotechnique institute journal, 2005,18 (4): 49-52).Summary is: after golden clock rattan methyl alcohol crude extract dry matter is dissolved with sterile purified water, be mixed with certain density mother liquor, get then the 1ml mother liquor join the 9ml fusion, be cooled in about about 50 ℃ potato dextrose agar (PDA) medium, fully shake up, pour into rapidly in the culture dish that diameter is 6cm, leave standstill and wait to condense, make and contain the toxic culture plate of PDA that methyl alcohol crude extract dry substance concentration is 10mg/ml.Be contrast if add sterile distilled water.All cultivating in the PDA medium in advance for the examination germ, is that the card punch of 0.5cm cuts the consistent bacterium piece of growth as inoculum at colony edge with the aperture.Inoculum is moved into the toxic culture plate of PDA central authorities.Put 25 ℃ of constant temperature culture.Measure colony diameter with the right-angled intersection method, and proofread and correct, calculate antibacterial percentage.
Bacteriostasis rate (%)=(contrast colony diameter-processing colony diameter)/(contrast colony diameter) * 100
3. result of the test
Gold clock rattan different parts methyl alcohol crude extract sees Table 1 to the inhibitory action test result of phytopathogen mycelial growth.
The methyl alcohol crude extract of table 1 gold clock rattan different parts is to the inhibitory action of phytopathogen mycelial growth
*
*Annotate: mensuration concentration is 10mg/ml; Colony diameter is 3 mean value ± S.E. that repeat
As seen from Table 1, under the mensuration concentration that contains golden clock rattan methyl alcohol crude extract dry matter 10mg/ml, the methyl alcohol crude extract of golden clock rattan different parts all shows bacteriostatic activity to the mycelial growth for the various plants pathogen of trying.Comparatively speaking, the bacteriostatic activity of the methyl alcohol crude extract at old rattan (containing cortex) position is higher than the tender rattan bacteriostatic activity of (containing blade).
In the phytopathogen for examination, the methyl alcohol crude extract of the old rattan of golden clock rattan (containing cortex) all is 100% to the bacteriostasis rate of peronophythora litchi and cucumber phytophthora, bacteriostasis rate to rice blast fungus also reaches 98.48%, inhibiting rate to Phomopsis citri is 72.34%, bacteriostatic activity to rhizoctonia and Luo Shi southern blight bacterium is respectively 65.40% and 63.16%, to tobacco leaf point mould, the inhibiting rate of Glorosprium musarum Cookeet Mass and tomato early blight bacterium is respectively 58.49%, 55.88% and 52.38%, to pythium spp, the bacteriostatic activity of pineapple black rot and withered germ of water-melon is respectively 45.90%, 36.67% and 33.68%.What the gold tender rattan of clock rattan (containing blade) methyl alcohol crude extract showed high bacteriostatic activity is to cucumber phytophthora root rot bacterium, and bacteriostasis rate is 83.33%, be to the peronophythora litchi bacterium secondly, and bacteriostasis rate is 62.27%, to other for the bacteriostasis rate that tries pathogen all less than 60%.
The methyl alcohol crude extract of embodiment 5 gold medal clock rattan plant different parts is to the bacteriostatic activity of plant pathogenic fungi, food mould and plant pathogenetic bacteria
1. for the examination pathogen
Plant pathogenic fungi: botrytis cinerea (Botrytis cinerea), citrus Penicillium notatum (Penicillium italicum).
Food mould: aspergillus niger (Aspergillus niger)
Plant pathogenetic bacteria: Xanthomonas oryzae (Xanthomonas oryzae pv.oryzae), bacterial wilt of tomato bacterium (Ralstonia solanacearum)
2. test method
Adopt inhibition zone method to measure the bacteriostatic activity of golden clock rattan plant different parts methyl alcohol crude extract to plant pathogenic fungi (tomato gray mould bacterium and citrus Penicillium notatum), food mould (aspergillus niger) and plant pathogenetic bacteria (Xanthomonas oryzae and bacterial wilt of tomato bacterium).Botrytis cinerea, citrus Penicillium notatum and aspergillus niger all are to use the PDA medium culture, and Xanthomonas oryzae and bacterial wilt of tomato bacterium are then cultivated with bacteria culture media.Wash fungal spore and bacterium lawn with sterile purified water, be mixed with certain density spore suspension and bacterial suspension respectively.Get 200 μ l hanging drops and be added on middle PDA culture medium flat plate or the bacteria culture media flat board, suspension is coated on the flat board uniformly with the bent glass bar of sterilization.Gold clock rattan methyl alcohol crude extract dry matter dissolves with sterile purified water, being mixed with concentration is: the soup that contains golden clock rattan methyl alcohol crude extract dry matter 100mg/ml, cut-off directly dips in soup for the filter paper of 0.5cm, the filter paper that will contain soup under the room temperature dries, then, the filter paper that will contain soup is placed on the culture plate that is coated with pathogen, puts in the 27 degree constant incubators and cultivates.The expression soup that inhibition zone occurs has bacteriostatic activity to the pathogen for examination; Measure the size of inhibition zone, and represent the power of bacteriostatic activity with the size of inhibition zone.
3. result of the test
Gold clock rattan different parts methyl alcohol crude extract sees Table 2 to the bacteriostatic activity of plant pathogenic fungi, food mould and plant pathogenetic bacteria.
The methyl alcohol crude extract of table 2 gold clock rattan different parts is to the inhibitory action of phytopathogen
*
*Annotate: mensuration concentration is 100mg/ml; Antibacterial circle diameter is 3 mean value ± S.E. that repeat; " +++"
The expression bacteriostatic activity is strong, " ++ " expression bacteriostatic activity is strong, "+" expression has bacteriostatic activity, the no bacteriostatic activity of "-" expression
As seen from Table 2, the methyl alcohol crude extract of golden clock rattan different parts all has stronger activity to botrytis cinerea and citrus pathogens penicillium; Then only there is tender rattan to show more weak bacteriostatic activity to aspergillus niger; Two plant species pathogenetic bacterias are also shown bacteriostatic activity.
From table 1 and table 2 as seen, the antibacterial activity of golden clock rattan plant extracts is composed suitable wide spectrum.
The methyl alcohol crude extract aqua of embodiment 6 gold medal clock rattan plant different parts is measured the control efficiency of cucumber downy mildew
1. for the examination pathogen
Bacterium of downy mildew of cucumber (Pseudoperonospora cubensis)
2. test method
Adopt leaf dish method to measure the control efficiency of the cucumber downy mildew disease that golden clock rattan plant methyl alcohol crude extract aqua causes bacterium of downy mildew of cucumber.Cucumber variety for examination is a Chang Chun Mi Ci.Get complete open cucumber leaves, get the leaf dish with the card punch of 1.5cm.Inoculate method (Pan Ruqian, the Gu Xixin of the preparation method of the sporangia suspension of using bacterium of downy mildew of cucumber with reference to Pan Ruqian.The cucumber different cultivars is to the resistance research [J] of downy mildew.Agricultural University Of South China's journal, 1993,14 (2): 61-67), the concentration of sporangia suspension is 10
5Individual sporangium/ml.Gold clock rattan methyl alcohol crude extract is dissolved with sterile purified water, and it is the aqua soup that contains golden clock rattan methyl alcohol crude extract dry matter 10mg/ml that adding auxiliary agent polysorbas20 (0.1%) is mixed with concentration.Add 5ml aqua soup in the culture dish of 9cm, put into 10 leaf dish then, drip the sporangia suspension of 1 10 μ l on every leaf dish.Put the following dark culturing of 24 degree 24 hours, and after cultivating 6～8 days under the illumination in 16 hours, checked incidence then, measure lesion area with the leaf area measuring instrument.Calculate disease index and control efficiency.If 1000 times of liquid of commercially available agricultural chemical 58% metalaxyl manganese-zinc wettable powder, promptly containing effective constituent concentration is that 580 μ g a.i./ml and clear water are treated to contrast.The grade scale of the state of an illness is as follows:
0 grade: no scab;
1 grade: lesion area accounts for the area of whole leaf dish less than 5%;
3 grades: lesion area accounts for the area of whole leaf dish between 6-25%;
5 grades: lesion area accounts for the area of whole leaf dish between 26-50%;
7 grades: lesion area accounts for the area of whole leaf dish greater than more than 51%
3. result of the test
Gold clock rattan plant different parts methyl alcohol crude extract aqua sees Table 3 to the control efficiency of cucumber downy mildew.
Table 3 gold clock rattan different parts methyl alcohol crude extract aqua is to the control efficiency of cucumber downy mildew
| Handle | Disease index | Control efficiency (%) |
| The gold old rattan of clock rattan (containing cortex) methyl alcohol crude extract aqua | 8.20 | 80.28 |
| The gold tender rattan of clock rattan (containing blade) methyl alcohol crude extract aqua | 16.25 | 60.93 |
| 58% metalaxyl-mn-zn WP (1000 times of liquid) | 9.19 | 77.90 |
| The clear water contrast | 41.59 |
Annotate: the concentration of treatment of Merremia boisiana extract aqua is to contain methyl alcohol crude extract dry matter 10mg/ml
By table 3 as seen, the methyl alcohol crude extract aqua of old rattan of golden clock rattan (containing cortex) and tender rattan (containing blade) all has control efficiency to cucumber downy mildew, and the control efficiency under 10mg/ml concentration is respectively 80.28% and 60.93%.Gold clock rattan old rattan methyl alcohol crude extract aqua is suitable to the drug effect of the control efficiency of cucumber downy mildew and commercially available agricultural chemical metalaxyl-mn-zn.
In addition, the methyl alcohol crude extract aqua of old rattan of golden clock rattan and tender rattan does not all have poisoning to cucumber leaves, and it is dark green that the blade of processing keeps.The result shows that golden clock rattan methanolic extract aqua is to plant safety.
From table 1, table 2 and table 3 as seen, except having the bacteriostatic activity of wide spectrum under exsomatizing, golden clock rattan plant extracts also has live body protection effect preferably.
The bacteriostatic activity of embodiment 7 gold medal clock rattan plant different organic solvents extracts
1. for the examination pathogen
Rice blast fungus (Magnaporthe grisea), peronophythora litchi bacterium (Peronophthora litchii), rhizoctonia (Rhizoctonia solani), cucumber phytophthora (Phytophthora melonis), Glorosprium musarum Cookeet Mass (Colletotrichum musae).
2. test method
Adopt the growth rate method of toxic medium to measure the inhibitory action of golden clock rattan plant extracts to the pathogen mycelial growth.Test method is identical with embodiment 4 described methods.Ligroin extraction, chloroform extract and ethyl acetate extract are diluted with water to desired concn then with a spot of acetone (1%v/v) dissolving earlier, and n-butanol extract is water dissolving and dilution directly.The final concentration of measuring is except that ethyl acetate extract is 5mg/ml, and other extract is 10mg/ml.
3. result of the test
The different organic solution extracts of gold clock rattan see Table 4 to the inhibitory action test result of phytopathogen mycelial growth.
As seen from Table 4, golden clock rattan different organic solvents extract is to the inhibitory action difference of pathogen.The distribution difference that shows the bacteriostatic activity material.Under 10mg/ml concentration, ligroin extraction is only to Glorosprium musarum Cookeet Mass inhibitory action a little, rice blast fungus, peronophythora litchi bacterium, cucumber phytophthora and rhizoctonia all there is not bacteriostasis, promote growth of pathogenic bacteria on the contrary, and promote to produce spore (particularly to rice blast fungus and Glorosprium musarum Cookeet Mass, data are unlisted); The activity of chloroform extract has improved, and to two kinds of lower funguses for examination, peronophythora litchi and cucumber phytophthora all have higher activity, and bacteriostasis rate is respectively 85.76% and 100%; The bacteriostatic activity of ethyl acetate extract is the highest, under 5mg/ml concentration, all is 100% to the bacteriostasis rate of rice blast fungus, peronophythora litchi and cucumber phytophthora; N-butanol extract is 100% to the bacteriostasis rate of rhizoctonia then, is higher than the bacteriostatic activity of methyl alcohol crude extract far away, and n-butanol extract all shows higher activity to two kinds of lower funguses simultaneously; Water extract also has certain bacteriostatic activity, and the bacteriostatic activity of cucumber phytophthora root rot bacterium and peronophythora litchi bacterium is respectively 60.40% and 54.33%.
Table 4 gold clock rattan different organic solvents extract is to the inhibitory action (mensuration concentration is 10mg/ml) of phytopathogen mycelial growth
*The mensuration concentration of ethyl acetate extract is 5mg/ml.
Embodiment 8 gold medal clock rattan plant ethyl acetate extracts are observed cucumber phytophthora root rot bacterium bacteriostasis mechanism
1. for the examination pathogen
Cucumber phytophthora (Phytophthora melonis)
2. test method
Golden clock rattan plant ethyl acetate extract is made the toxic flat board (because under 1000 μ g/ml, suppressing mycelial growth fully) of 500 μ g/ml, on toxic flat board, cultivate cucumber phytophthora root rot bacterium, do control experiment picking mycelia simultaneously, the form of observation mycelia under light microscope.
3. result of the test
The bacterium colony of cucumber phytophthora root rot bacterium on the toxic flat board of golden clock rattan plant ethyl acetate extract is more sparse, and bacterium colony is unusual.Under light microscope, the obvious form deformity of visible mycelia: show as branch and increase, mycelia tubbiness, branch end expand etc.
Embodiment 9 gold medal clock rattan plant n-butanol extract aquas are measured the live body control efficiency of Rhizoctonia solani Kuhn
1. for the examination pathogen
Rhizoctonia solani Kuhn (Rhizoctonia solani) merges group GA-1
2. test method
Adopt the high Ban Shi excised leaf method of improvement to measure the live body control efficiency of golden clock rattan plant n-butanol extract aqua to Rhizoctonia solani Kuhn.As excised leaf, because the French beans blade is susceptible, and blade area is bigger with the French beans blade.Golden clock rattan n-butanol extract is dissolved with aqua sterilisa, and add 0.1% auxiliary agent polysorbas20, be mixed with and contain the aqua soup that the n-butanol extract dry matter is 1000 μ g/ml concentration, be coated on then on the French beans blade that exsomatizes.Naturally after drying, at a blade central authorities inoculation Rhizoctonia solani Kuhn bacterium piece (0.5cm diameter), 10 blades are inoculated in every processing altogether, establish that commercially available agricultural chemical 50% carbendazol wettable powder (1000 μ g/ml) and 25% Amici reach suspension (250 μ g/ml) and clear water is treated to contrast.Each is handled 25 and spends the cultivation 4 days of preserving moisture, and checks the size of lesion area, calculates control efficiency.
3. result of the test
Gold clock rattan plant n-butanol extract aqua the results are shown in Table 5 to the live body control efficiency of Rhizoctonia solani Kuhn.
Table 5 gold clock rattan plant n-butanol extract aqua is to the live body control efficiency of rice sheath blight disease
| Handle | Lesion area (cm 2) | Control efficiency (%) |
| Gold clock rattan plant n-butanol extract aqua (1000 μ g/ml) | 1.67±0.13 | 88.32 |
| 50% carbendazol wettable powder (1000 μ g/ml) | 1.45±0.25 | 89.86 |
| 25% Amici reaches suspension (250 μ g/ml) | 2.05±0.09 | 85.66 |
| The clear water contrast | 14.30±2.31 |
By table 5 as seen; after adding certain auxiliary agent; gold clock rattan n-butanol extract is under 1000 μ g/ml concentration; bacteriostasis to Rhizoctonia solani Kuhn is very strong; can effectively protect the pathogenic effects of sheath blight fungus to the French beans blade; control efficiency reaches 88.32%, and effect slightly is better than commercially available agricultural chemical Fluoxastrobin (25% Amici reaches the trade name that suspension is Syngenta Co.,Ltd), and suitable with the preventive effect of the carbendazim of same concentration.
Do not observe any bad poisoning phenomenon.The result shows that golden clock rattan n-butanol extract aqua is to plant safety.
Embodiment 10 gold medal clock rattan ethyl acetate extracts are to the mode of action and the mechanism of peronophythora litchi
1. for the examination pathogen
Peronophythora litchi bacterium (Peronophthora litchii)
2. test method
2.1 influence to the sprouting of peronophythora litchi sporangium
Adopt the spore germination method to measure the influence that golden clock rattan ethyl acetate extract is sprouted the peronophythora litchi sporangium.Extract then with the sterile water dilution, is mixed with the soup of series concentration earlier with small amount of acetone (1%v/v) dissolving.Small amount of impurities disturbance-observer in the solution can filter to remove impurity solution with filter paper earlier.Medicine liquid droplet is added in the peronophythora litchi bacterium sporangia suspension, draw 50 μ l and contain the sporangia suspension of soup on concave slide, put Celsius 24 and spend the cultivation 6 hours of preserving moisture,, measure the minimum inhibitory concentration (MIC) of soup then in the sporangial sprouting situation of test under microscope.Sporangium is sprouted (it is empty that sporangium becomes) indirectly and sprouting is all counted in direct germination (sporangium generation germ tube).Establish 1% acetone and aqua sterilisa respectively and be contrast.
2.2 to the zoospore motility and to the influence of zoospore
Golden clock rattan ethyl acetate extract is diluted to the series concentration soup as stated above, and medicine liquid droplet is added in the zoospore suspension, at microscopically observation variable concentrations soup to the motility of zoospore and to the influence of zoospore.
2.3 influence to the spore germination of stopping
Adopt the spore germination method to measure golden clock rattan ethyl acetate extract to the stop influence of spore germination of peronophythora litchi.Processing method is the same.At 100 spores that stop of microscopically casual inspection, the statistics germination rate, and proofread and correct, calculate and suppress germination rate.
3. result of the test
3.1 influence to the sporangium sprouting
Gold clock rattan ethyl acetate extract has had strong inhibitory effects to the sporangial sprouting of peronophythora litchi bacterium.The minimum inhibitory concentration of soup is 500 μ g/ml, under this concentration, suppresses germination rate and reaches 100%.
3.2 to the motility of zoospore and to the effect of zoospore
Gold clock rattan ethyl acetate extract can make zoospore stop to move about under 500 μ g/ml concentration immediately; Under 250 μ g/ml concentration, most of zoospores stop to move about in 10 minutes.Under 50 μ g/ml concentration, moving about of zoospore is unaffected substantially, and zoospore can normally move about.We observe a very special phenomenon: under 250 μ g/ml concentration, zoospore is after stopping to move about, and most of zoospores pop and break in 30 minutes, are dissolved fully.
3.3 effect to the spore germination of stopping
Under 500 μ g/ml concentration, the sprouting of 98% the spore that stops is suppressed; Under 50 μ g/ml concentration, there is the sprouting of 73% the spore that stops to be suppressed.Show very strong bactericidal activity.
The present patent application people has compared more existing new bactericide, such as commercial dimethomorph of nineteen ninety (dimethomorph) and the Fluoxastrobin that began commercial respiratory chain complex III inhibitor (claiming the strobilurins series bactericidal agent again) in 1996 the mode of action to the different phase of peronophythora litchi bacterium history of life, the former has had strong inhibitory effects to the sprouting of the spore that stops, but the motility of zoospore is not influenced; The latter then has strong effect to the motility of zoospore and the sprouting of the spore that stops.Experimental results show that the mode of action of golden clock rattan ethyl acetate extract has some characteristics of above two kinds of bactericide concurrently, each vegetative stage of pathogen is all had very strong activity; And unique mode is arranged: can make the zoospore explosion.This is a very unique mechanism of action, is worth further research.
Claims (2)
1, the application of Merremia boisiana extract in the preparation bactericide, wherein, described Merremia boisiana extract is to adopt chloroform, ethyl acetate, methyl alcohol, n-butanol or water, by the extract that the dipping bubble extracts under the room temperature, percolation extracts or decocting method extracts.
2, application as claimed in claim 1, what it is characterized in that described golden clock rattan employing is root, stem and/or the blade of golden clock rattan plant.
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| CN111543326B (en) * | 2020-06-09 | 2022-11-18 | 海南大学 | Asexual propagation method for Stephania delavayi Franch |
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| EP0277736A2 (en) * | 1987-02-03 | 1988-08-10 | THE ROYAL INSTITUTION FOR THE ADVANCEMENT OF LEARNING (McGILL UNIVERSITY) | Fungal herbicides |
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