CN102763662B - Medium for separating Peronophythora Litchi Chen ex Ko et al - Google Patents
Medium for separating Peronophythora Litchi Chen ex Ko et al Download PDFInfo
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Abstract
The invention discloses a medium for separating Peronophythora Litchi Chen ex Ko et al. The medium is I or II shown as below: I. a medium obtained by adding bactericides comprising Difenoconazole, carbendazim, rifampicin and penicillin into a white kidney bean medium; and II. a medium obtained by adding bactericides comprising Difenoconazole, prochloraz, rifampicin and penicillin into a white kidney bean medium. The white kidney bean selective medium provided by the invention is low-cost and easily prepared, and has good inhibition effect on bacteria. Experiments have proved that the method for separating Peronophythora Litchi Chen ex Ko et al by the white kidney bean selective medium provided by the invention solves problems of severe pollution caused by non-target fungi and bacteria in a separation process of plant pathogen oomycetes like Peronophythora Litchi Chen ex Ko et al, and has advantages of high separation efficiency, reliability, time saving and labor saving.
Description
Technical field
The present invention relates to a kind of medium for separating of the Peronophythora Litchii germ.
Background technology
Lichee (Litchi chinensis Sonn.) is the famous good fruit in the south of the Five Ridges, 18 °～31 ° of north latitude, plantation is all arranged, and distributes and spreads all over 8 provinces and regions such as Hainan, Guangxi, Yunnan, Fujian, Guizhou, Sichuan, Guangdong, Taiwan.Within 2008, national cultivated area is approximately 9,000,000 mu, surpasses the approximately lichee of 8,200,000 mu, becomes the tropical fruit (tree) of cultivated area maximum.The Yield of Litchi of China surpasses 60% of global output.Peronophythora litchi is a kind of Major Diseases of lichee, and this disease can endanger the Hua Heguo of lichee, and the florescence morbidity, cause a large amount of withered flowers, and the young fruit period morbidity, cause shedding, and the maturing stage morbidity, cause fruit rot.In the fresh fruit transportation and sale phase, this sick continuation is developed, and also has a strong impact on storing and the export trade of fresh fruit, and then has affected the sustainable stable development of lichee industry.Chemical control is to prevent and treat at present one of effective method of this disease.
Peronophythora Litchii germ (Peronophythora Litchi Chen ex Ko et al) survives the winter in sick leaf, sick fruit and soil with mycelium and egg spore, is the first pathogeny that infects in next year.The end of spring and the beginning of summer in next year produces sporangium, with wind and rain, propagates, and the zoospore that sporangium produces is invaded host's blade and fruit is caused harm.For the Peronophythora Litchii germ is carried out to correlative study, and the bacterium amount of surviving the winter and the field incidence of investigation pathogen, carry out the prediction of disease, the Peronophythora Litchii germ that obtains purifying by separation is necessary.
Conventional pathogenicbacteria separation method is that use 75% ethanol and the 0.1% mercuric chloride liquid surface sterilization that " planting the disease research method " introduced processed.In real work, the sample separated in laboratory is not often fresh sample, it is the invalid body of depositing certain hour after Field sampling, in these litchi sickness tissues just inevitably usually with other miscellaneous bacterias, while carrying out the chorista pathogen according to a conventional method, because the growth of these miscellaneous bacterias is rapid than the Peronophythora Litchii germ, the mycelia of miscellaneous bacteria often covers the Peronophythora Litchii germ, and the bacterium of Fast Growth also can suppress the growth of Peronophythora Litchii germ.
In tradition pathogenicbacteria separation method, while utilizing ordinary culture medium to separate Peronophythora Litchii from diseased tissues, if the diseased tissues of collection is not sterilized or disinfecting time is short, cause while cultivating living contaminants is arranged, do not reach the purpose of separation and purification.If disinfecting time is oversize, often, when killing miscellaneous bacteria, also can kill the pathogen that will separate, cause the pathogenicbacteria separation rate often not too high.
Summary of the invention
An object of the present invention is to provide a kind of bactericide, for separating of medium and the method for separating Peronophythora Litchii germ (Peronophythora Litchi Chen ex Ko et al) of Peronophythora Litchii germ (Peronophythora Litchi Chen ex Ko et al).
Bactericide provided by the present invention, its active component is following 1) or 2) shown in:
1) by Difenoconazole, carbendazim, rifampin and penicillin, formed;
2) by Difenoconazole, Prochloraz, rifampin and penicillin, formed.
Above-mentioned bactericide, described 1) shown in bactericide, the mass fraction of described Difenoconazole, carbendazim, rifampin and penicillin is than being (10-20): (10-50): (50-100): (50-100), be specially 10:10:50:50, or 15:20:70:70, or 20:50:100:100;
Above-mentioned bactericide, described 2) shown in bactericide, the mass fraction of described Difenoconazole, Prochloraz, rifampin and penicillin is than being (10-20): (10-50): (50-100): (50-100), be specially 10:10:50:50, or 15:20:70:70, or 20:50:100:100.
Medium for separating of Peronophythora Litchii germ (Peronophythora Litchi Chen ex Ko et al) provided by the present invention, shown in following I or II:
I, to adding described in claim 1 or 21 in the Semen Phaseoli Vulgaris medium) shown in bactericide obtain;
II, to adding described in claim 1 or 22 in the Semen Phaseoli Vulgaris medium) shown in bactericide obtain.
Shown in above-mentioned I, in medium, the concentration of described Difenoconazole in described Semen Phaseoli Vulgaris medium is 10 μ g/mL-20 μ g/mL, is specially 10 μ g/mL, 15 μ g/mL or 20 μ g/mL; The concentration of described carbendazim in described Semen Phaseoli Vulgaris medium is 10 μ g/mL-50 μ g/mL, is specially 10 μ g/mL, 20 μ g/mL or 50 μ g/mL; The concentration of described rifampin in described Semen Phaseoli Vulgaris medium is 50 μ g/mL-100 μ g/mL, is specially 50 μ g/mL, 70 μ g/mL or 100 μ g/mL; The concentration of described penicillin in described Semen Phaseoli Vulgaris medium is 50 μ g/mL-100 μ g/mL, is specially 50 μ g/mL, 70 μ g/mL or 100 μ g/mL;
Shown in above-mentioned II, in medium, the concentration of described Difenoconazole in described Semen Phaseoli Vulgaris medium is 10 μ g/mL-20 μ g/mL, is specially 10 μ g/mL, 15 μ g/mL or 20 μ g/mL; The concentration of described Prochloraz in described Semen Phaseoli Vulgaris medium is 10 μ g/mL-50 μ g/mL, is specially 10 μ g/mL, 20 μ g/mL or 50 μ g/mL; The concentration of described rifampin in described Semen Phaseoli Vulgaris medium is 50 μ g/mL-100 μ g/mL, is specially 50 μ g/mL, 70 μ g/mL or 100 μ g/mL; The concentration of described penicillin in described Semen Phaseoli Vulgaris medium is 50 μ g/mL-100 μ g/mL, is specially 50 μ g/mL, 70 μ g/mL or 100 μ g/mL.
The application of above-mentioned medium in separating Peronophythora Litchii germ (Peronophythora Litchi Chen ex Ko et al) also belongs to protection scope of the present invention.
The method of separation Peronophythora Litchii germ provided by the present invention (Peronophythora Litchi Chen ex Ko et al), comprise the steps: to obtain the Peronophythora Litchii germ with above-mentioned arbitrary described medium culture sample; Described sample is blade or the fruit of the plant of infection Peronophythora Litchii germ.
Semen Phaseoli Vulgaris selective medium of the present invention is with low cost, simple for production, and bacterium is had to extraordinary inhibitory action.Experiment showed, the method for separating Peronophythora Litchii with Semen Phaseoli Vulgaris selective medium of the present invention, solved the with serious pollution problem of various non-target fungus and bacterium in the pathogenic oomycetes separation process such as Peronophythora Litchii; The method separative efficiency is high, reliable, save time, laborsaving.
The accompanying drawing explanation
Fig. 1 is the inhibitory action of three kinds of antibiotic to the plant leaf surface bacteria.
Fig. 2 is the bacteriostasises of three kinds of antibiotic to the pericarp surface.
The symptom that Fig. 3 is peronophythora litchi (being photographed Beiliu City, Guangxi)
Embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Carbendazim active compound is purchased from Guangxin, Anhui Nong Hua limited company (Guangxin, Anhui Nong Hua Group Co.,Ltd), the chemical name of its main active material is N-(2-benzimidazole base)-methyl carbamate, adopted name is carbendazim, molecular formula: C9H9N3O2.In this medicine, the quality percentage composition of main active material is 98.9 ﹪.
Difenoconazole technical material is purchased from HangZhou YuLong Chemical Engineering Co., Ltd.The chemical name of its main active material is suitable, the chloro-4-[4-methyl-2-1H-1 of trans-3-, 2,4-triazol-1-yl methyl)-1, the mute pentane of 3-bis--2-yl) phenyl 4-chlorphenyl ether (suitable, inverse proportion is about 45:55), adopted name is Difenoconazole.In this medicine, the quality percentage composition of main active material is 96 ﹪.
The former medicine of Prochloraz (being again Prochloraz) is purchased from Hainan power intelligence biotechnology Co., Ltd, the chemical name of its main active material is N-propyl group-N-[2-(2,4,6-Trichlorophenoxy) ethyl]-imidazoles-1-formamide, molecular formula: C15H16Cl3N3O2.In this medicine, the quality percentage composition of Prochloraz is 97 ﹪.
Penicillin, rifampin, chloramphenicol, streptomycin, neomycinsulphate and quadracycline are all purchased from Beijing Tuo Yingfang Science and Technology Ltd..
Three kinds of fungistats of embodiment 1, variable concentrations and the six kinds of antibiotic inhibitory action to Peronophythora Litchii
One, three of variable concentrations kinds of fungistats inhibitory action to Peronophythora Litchii
The solvent that the dimethyl formamide of take is carbendazim, take the solvent that the third bronze medal or methyl alcohol is Difenoconazole and Prochloraz, prepares respectively three kinds of medicament mother liquor 10ml of 5000 μ g/mL, and the weighed amount of three kinds of medicaments is followed successively by 50.6mg, 52.0mg and 51.5mg; These three kinds of mother liquors are prepared respectively to the carbendazim that series concentration is 50 μ g/mL, 20 μ g/mL and 10 μ g/mL, the band poison Semen Phaseoli Vulgaris flat board of Prochloraz and Difenoconazole, the flat board that is added with solvent of take is contrast; Then adopt the mycelial growth rate method to measure the susceptibility of Peronophythora Litchii to three kinds of fungistats.By the Peronophythora Litchii bacterial strain on the Semen Phaseoli Vulgaris flat board in 25 ℃ of preculture 7d, the card punch that is 5mm with diameter is beaten and is got the bacterium cake on the same circumference of colony edge, by pure culture biscuits involvng inoculation to the dull and stereotyped central authorities of the Semen Phaseoli Vulgaris band poison that contains respectively three kinds of medicine series concentration (each concentration is containing the equivalent organic solvent), in 25 ℃ of lower dark culturing, measure each bacterium colony of processing by the right-angled intersection method after 7d and increase diameter (colony diameter deducts bacterium cake diameter 5mm), every processing repeats 3 times; Finally according to following formula, obtain the inhibition percentage (%) of each drug concentration to mycelial growth, inhibiting rate (%)=[(the contrast bacterium colony increases diameter-processing bacterium colony and increases diameter)/(the contrast bacterium colony increases diameter)] * 100.
Measured the virulence to Peronophythora Litchii for three kinds of fungistats of examination, result is as shown in table 1, and the carbendazim of variable concentrations all lower than 10%, therefore can adopt the carbendazim of 10-50 μ g/mL as selective pesticides to the inhibiting rate of Peronophythora Litchii; The medicament of Difenoconazole 50 μ g/mL concentration to the inhibiting rate of Peronophythora Litchii in 20% left and right, the Difenoconazole of 20 μ g/mL concentration has facilitation on the contrary to the growth of Peronophythora Litchii, the concentration of 10 μ g/mL does not have inhibitory action to Peronophythora Litchii yet, therefore, can adopt the Difenoconazole of 10-20 μ g/mL concentration as selective pesticides; The medicament of Prochloraz 50 μ g/mL concentration has facilitation to Peronophythora Litchii, and inhibiting rate is-68.7%.The Prochloraz of 20 and 10 μ g/mL concentration does not all have inhibitory action to Peronophythora Litchii, so can adopt the selective pesticides of Prochloraz for using in the separation Peronophythora Litchii of 10-50 μ g/mL yet.
The inhibitory action of three kinds of fungistats of table 1 to Peronophythora Litchii
Two, six of variable concentrations kinds of antibiotic inhibitory action to Peronophythora Litchii
With aqua sterilisa, six kinds of antibiotic all are mixed with to 5 * 10
4the mother liquor of μ g/mL, six kinds of mother liquors are mixed with respectively to rifampin, penicillin and the chloramphenicol that concentration is 100 μ g/mL, 50 μ g/mL, the streptomycin of 50 μ g/mL, the quadracycline of 30 μ g/mL and 20 μ g/mL, the pastille Semen Phaseoli Vulgaris flat board of the neomycinsulphate of 100 μ g/mL, 50 μ g/mL and 30 μ g/mL, the flat board that is added with sterile water of take is contrast; Then adopt the mycelial growth rate method to measure Peronophythora Litchii to six kinds of antibiotic susceptibility.By the Peronophythora Litchii bacterial strain on the Semen Phaseoli Vulgaris flat board in 25 ℃ of preculture 7d, the card punch that is 5mm with diameter is beaten and is got the bacterium cake on the same circumference of colony edge, by pure culture biscuits involvng inoculation to the dull and stereotyped central authorities of the Semen Phaseoli Vulgaris band poison that contains respectively six kinds of medicine series concentration (each concentration is containing the equivalent sterile water), in 25 ℃ of lower dark culturing, measure each bacterium colony of processing by the right-angled intersection method after 7d and increase diameter (colony diameter deducts bacterium cake diameter 5mm), every processing repeats 3 times; Finally according to following formula, obtain the inhibition percentage (%) of each drug concentration to mycelial growth, inhibiting rate (%)=[(the contrast bacterium colony increases diameter-processing bacterium colony and increases diameter)/(the contrast bacterium colony increases diameter)] * 100.
Measured the inhibitory action of six kinds of antibiotic of variable concentrations to Peronophythora Litchii, the results are shown in Table 2, the penicillin that concentration is 100 and 50 μ g/mL, rifampin and chloramphenicol all do not have obvious inhibitory action to Peronophythora Litchii; And concentration is 30 and 20 μ g/mL quadracyclines, Peronophythora Litchii is all had to very strong inhibitory action, inhibiting rate can reach 80%; The neomycinsulphate medicament that concentration is 30 μ g/mL, 50 μ g/mL and 100 μ g/mL all has very strong inhibitory action to Peronophythora Litchii, the highlyest can reach 66.9%.Streptomycin has very strong inhibitory action for Peronophythora Litchii, can not use as selective pesticides.In sum, tentatively clearly can use penicillin, rifampin and the chloramphenicol of 50-100 μ g/mL as the antibiotic composition in separation and purification Peronophythora Litchii selective medium.And intend further measuring the inhibitory action of three kinds of antibiotic to the bacterium of carrying with soil bacteria and capsicum tissue in environment.
Table 2 is used the inhibitory action of six kinds of antibiotic to Peronophythora Litchii
Embodiment 2, the three kind of antibiotic inhibitory action to bacterium in environment
This test is to selected three kinds of antibiotic detections on the basis of embodiment 1 result.Whether check during to the antibiotic concentration of Peronophythora Litchii unrestraint effect, can effectively suppress the bacterium in environment when employing.For the detection of bacterium in environment, mainly for the bacterium from plant leaf and morbidity fruit pericarp, this is to be mainly disease leaf, sick decisive and resolute fixed according to the separated part of Peronophythora Litchii germ.At first, be the inhibitory action to the Litchi Leaves surface bacteria: 5 point samplings are taked 10 Litchi Leaves at random, shred and put into the 50ml centrifuge tube, add the 15ml aqua sterilisa, 200r/min vibration 2 minutes, cross leaching filtrate, use sterile water dilution 100 *, get respectively on rifampin, penicillin and the chloramphenicol band poison Semen Phaseoli Vulgaris flat board that 100 μ l are coated on 100 μ g/mL, 50 μ g/mL, take and be not with malicious flat board as contrast, 3 repetitions of every processing, observe its bacteriostasis after 2 days; Then get 5 sick fruits from the lichee epidemic disease serious field of falling ill, stripping pericarp puts into triangular flask and adds the 100ml aqua sterilisa, the 200r/min 20min that vibrates, get on the band poison Semen Phaseoli Vulgaris flat board of rifampin, penicillin and chloramphenicol that supernatant 100 μ l are coated on 100 μ g/mL, 50 μ g/mL, take and be not with malicious flat board as contrast, 3 repetitions of every processing, within 2 days, observe afterwards top ne ar and clump count the number, with the judgement antibiotic to the inhibitory action of bacterium in environment.
This research is on the basis of table 2 result, by initial option can with antibiotic carried out the test of its inhibitory action to bacterium on blade and fruit, the result of two researchs is consistent, from Fig. 1 and Fig. 2, can find out, compared with the control, the bacteriostatic activity of penicillin and rifampin is stronger, can substantially reach antibacterial effect, diversity due to bacterial species in environment, penicillin mainly has stronger inhibitory action to gram-positive bacteria, so can see in Fig. 1 and 2 a little fragmentary bacterium colony is arranged, judge that it is Gram-negative bacteria.And the rifampin fungicidal spectrum is wide, can see only having a little fungus colony in flat board from Fig. 1 and 2; And Fig. 1 and 2 shows, the chloramphenicol bacteriostatic activity is poor, and when its concentration reaches 100 μ g/mL, compared with the control, fungistatic effect is not obvious.Therefore, rifampin can coordinate penicillin to use, to reach better bacteriostasis;
In sum, the principle that the present invention screens medicament is that the Peronophythora Litchii inhibiting rate is less than under 20% prerequisite, selects as far as possible the medicament of high concentration, to improve the bacteriostatic activity to non-object bacteria.Can find out according to above all result of the tests, be applied to the selective medium of the disposable separation peronophythora litchi from field, the fungistat that can select is carbendazim, Difenoconazole and Prochloraz, and concentration is 10-50 μ g/mL; The antibiotics that can select is penicillin and rifampin, and concentration all can adopt 50-100 μ g/mL.
Embodiment 3, for separating of the Semen Phaseoli Vulgaris of Peronophythora Litchii germ, select medium
One, separating method
In 2009,2010 years from Fujian, Guangdong and Hainan gathers sick sample in the serious area of peronophythora litchi morbidity, for Peronophythora Litchii, separate.The incidence of field Phytophthora capsici as shown in Figure 2.When A. blade is injured, the initial stage is small brown specks, after progressively expand as irregular yellowish-brown scab, withered dead, cause a large amount of fallen leaves.When B. fruit is injured, scab is many to start to occur from base of fruit, and brown is irregular shape, without limbus, grows the mould layer of white as frost when moist.
The separating method of Peronophythora Litchii carries out as follows: select different fields at areal, gather peronophythora litchi sample (blade, fruit) according to the local morbidity order of severity and separated.
Separating step: use clear water to clean out incidence of leaf and fully dry afterwards, the fritter tissue that cuts the strong intersection of disease is seeded on selective medium of the present invention, or be that sporangium is chosen to selectivity Semen Phaseoli Vulgaris medium of the present invention by the white frost layer on fresh sick fruit, 25 ℃ of dark culturing, to there being mycelia to grow; As not obvious as the white frost layer on the disease fruit, can be placed on 4 ℃ of refrigerators and cultivate in 25 ℃ of incubators after 1 day, occur separating according to the method described above immediately after frost layer.The picking mycelia is transferred on common white kidney bean medium and cultivates, and ware to be covered with is placed on 25 ℃ of lower alternation of light and darkness and cultivates about 7 days, induces it to produce sporangium; Add aqua sterilisa in culture dish, be placed on Zoospore liberation after 15 ℃ of light treatment 60min; Choose single zoospore and be placed on common white kidney bean medium and cultivate, to growing bacterium colony, obtain Peronophythora Litchii.The Peronophythora Litchii obtained is identified.Real Peronophythora Litchii bacterium colony is kept on the PDA test tube slant, adds appropriate sterilizing paraffin oil and be placed under room temperature and save backup.
With the evaluation of existing Peronophythora Litchii germ, identified.
Two, separate the medium used:
(1) Semen Phaseoli Vulgaris medium: white clouds bean powder 60g, agar powder 12.5g, adding distil water is settled to 1000mL, high pressure steam sterilization, 121 ° of C, 20min.
(2) Semen Phaseoli Vulgaris is selected medium
Blade or fruit are all used to following medium.
Following medium is not being used in the zone of carbendazim generation resistance:
Semen Phaseoli Vulgaris is selected the medium I: add Difenoconazole, carbendazim, rifampin and penicillin in uncooled Semen Phaseoli Vulgaris medium, and be settled to 1 liter with uncooled Semen Phaseoli Vulgaris medium; It is 15 μ g/mL that Difenoconazole is selected the concentration in the medium I at Semen Phaseoli Vulgaris, it is 20 μ g/mL that carbendazim is selected the concentration in the medium I at Semen Phaseoli Vulgaris, it is 70 μ g/mL that rifampin is selected the concentration in the medium I at Semen Phaseoli Vulgaris, and it is 70 μ g/mL that penicillin is selected the concentration in the medium I at Semen Phaseoli Vulgaris.
Semen Phaseoli Vulgaris is selected the medium II: with Semen Phaseoli Vulgaris, select the medium I basic identical, difference is: it is 10 μ g/mL that Difenoconazole is selected the concentration in the medium II at Semen Phaseoli Vulgaris, it is 10 μ g/mL that carbendazim is selected the concentration in the medium II at Semen Phaseoli Vulgaris, it is 50 μ g/mL that rifampin is selected the concentration in the medium II at Semen Phaseoli Vulgaris, and it is 50 μ g/mL that penicillin is selected the concentration in the medium II at Semen Phaseoli Vulgaris.
Semen Phaseoli Vulgaris is selected the medium III: with Semen Phaseoli Vulgaris, select the medium I basic identical, difference is: it is 20 μ g/mL that Difenoconazole is selected the concentration in the medium III at Semen Phaseoli Vulgaris, it is 50 μ g/mL that carbendazim is selected the concentration in the medium III at Semen Phaseoli Vulgaris, it is 100 μ g/mL that rifampin is selected the concentration in the medium III at Semen Phaseoli Vulgaris, and it is 100 μ g/mL that penicillin is selected the concentration in the medium III at Semen Phaseoli Vulgaris.
Use following medium in the zone that carbendazim is produced to resistance:
Semen Phaseoli Vulgaris is selected the medium I: add Difenoconazole, Prochloraz, rifampin and penicillin in uncooled Semen Phaseoli Vulgaris medium, and be settled to 1 liter with uncooled Semen Phaseoli Vulgaris medium; It is 15 μ g/mL that Difenoconazole is selected the concentration in the medium I at Semen Phaseoli Vulgaris, it is 20 μ g/mL that Prochloraz is selected the concentration in the medium I at Semen Phaseoli Vulgaris, it is 70 μ g/mL that rifampin is selected the concentration in the medium I at Semen Phaseoli Vulgaris, and it is 70 μ g/mL that penicillin is selected the concentration in the medium I at Semen Phaseoli Vulgaris.
Semen Phaseoli Vulgaris is selected the medium II: with Semen Phaseoli Vulgaris, select the medium I basic identical, difference is: it is 10 μ g/mL that Difenoconazole is selected the concentration in the medium II at Semen Phaseoli Vulgaris, it is 10 μ g/mL that Prochloraz is selected the concentration in the medium II at Semen Phaseoli Vulgaris, it is 50 μ g/mL that rifampin is selected the concentration in the medium II at Semen Phaseoli Vulgaris, and it is 50 μ g/mL that penicillin is selected the concentration in the medium II at Semen Phaseoli Vulgaris.
Semen Phaseoli Vulgaris is selected the medium III: with Semen Phaseoli Vulgaris, select the medium I basic identical, difference is: it is 20 μ g/mL that Difenoconazole is selected the concentration in the medium III at Semen Phaseoli Vulgaris, it is 50 μ g/mL that Prochloraz is selected the concentration in the medium III at Semen Phaseoli Vulgaris, it is 100 μ g/mL that rifampin is selected the concentration in the medium III at Semen Phaseoli Vulgaris, and it is 100 μ g/mL that penicillin is selected the concentration in the medium III at Semen Phaseoli Vulgaris.
Three, Isolation and Identification result
In the process of carrying out as above each separation, Semen Phaseoli Vulgaris selects all do not have miscellaneous bacteria to grow in medium, only has the Peronophythora Litchii germ to grow, and illustrates that Semen Phaseoli Vulgaris selection medium of the present invention has suppressed varied bacteria growing, makes the separation of Peronophythora Litchii germ easier.The inventive method is seeded to selective medium pathological tissues to there being the peronophythora litchi filament length to go out from starting, and generally only needs 3-4 days, has greatly shortened disengaging time, has improved separative efficiency.Disengaging time shortens, and is mainly because selection medium of the present invention has effectively suppressed the growth of other fungus and bacterium, makes the Peronophythora Litchii germ become dominant microflora in medium, and growth rate improves, thereby has accelerated the separation process.
From the serious regional diseased tissues of peronophythora litchi morbidity of above-mentioned provinces and cities, be divided into from obtaining 68 strain strain Peronophythora Litchii germs.All through identifying, and be the Peronophythora Litchii germ.
The present invention is directed to and be difficult at present directly separating the Peronophythora Litchii germ that obtains purifying from invalid body, and the process of traditional pathogenicbacteria separation purification process is loaded down with trivial details, disinfecting time is difficult to control, low and the existing method of separation rate is owing to adopting the little and inappropriate problem of drug concentration of medicament disinfecting spectral limit, having developed can the disposable microbicide compositions that separates Peronophythora Litchii from field, difference according to Peronophythora Litchii separated part and the regional dispenser history of sampling, the microbicide compositions of separate cultivating for Peronophythora Litchii be take fungistat Difenoconazole and bacterial inhibitor rifampin as main, other fungistat added and bacterial inhibitor (antibiotic) can have different combinations.Selectable fungistat is Prochloraz or carbendazim, and concentration is 10-50 μ g/mL; The antibiotics bacterial inhibitor that can select is penicillin, and concentration is 50-100 μ g/mL.
Claims (6)
1. a bactericide, its active component is following 1) or 2) shown in:
1) by Difenoconazole, carbendazim, rifampin and penicillin, formed; The mass fraction of described Difenoconazole, carbendazim, rifampin and penicillin is than being (10-20): (10-50): (50-100): (50-100);
2) by Difenoconazole, Prochloraz, rifampin and penicillin, formed; The mass fraction of described Difenoconazole, Prochloraz, rifampin and penicillin is than being (10-20): (10-50): (50-100): (50-100).
2. bactericide according to claim 1, it is characterized in that: described 1) in bactericide, the mass fraction of described Difenoconazole, carbendazim, rifampin and penicillin is than being 10:10:50:50, or 15:20:70:70, or 20:50:100:100;
Described 2) shown in, in bactericide, the mass fraction of described Difenoconazole, Prochloraz, rifampin and penicillin is than being 10:10:50:50, or 15:20:70:70, or 20:50:100:100.
3. the medium for separating of Peronophythora Litchii germ (Peronophythora Litchi Chen ex Ko et a1), shown in following I or II:
I, to adding described in claim 1 or 21 in the Semen Phaseoli Vulgaris medium) shown in bactericide obtain; The concentration of described Difenoconazole in described Semen Phaseoli Vulgaris medium is 10 μ g/mL-20 μ g/mL; The concentration of described carbendazim in described Semen Phaseoli Vulgaris medium is 10 μ g/mL-50 μ g/mL; The concentration of described rifampin in described Semen Phaseoli Vulgaris medium is 50 μ g/mL-100 μ g/mL; The concentration of described penicillin in described Semen Phaseoli Vulgaris medium is 50 μ g/mL-100 μ g/mL;
II, to adding described in claim 1 or 22 in the Semen Phaseoli Vulgaris medium) shown in bactericide obtain; The concentration of described Difenoconazole in described Semen Phaseoli Vulgaris medium is 10 μ g/mL-20 μ g/mL; The concentration of described Prochloraz in described Semen Phaseoli Vulgaris medium is 10 μ g/mL-50 μ g/mL; The concentration of described rifampin in described Semen Phaseoli Vulgaris medium is 50 μ g/mL-100 μ g/mL; The concentration of described penicillin in described Semen Phaseoli Vulgaris medium is 50 μ g/mL-100 μ g/mL.
4. medium according to claim 3, it is characterized in that: shown in described I, in medium, the concentration of described Difenoconazole in described Semen Phaseoli Vulgaris medium is; 10 μ g/mL, 15 μ g/mL or 20 μ g/mL; The concentration of described carbendazim in described Semen Phaseoli Vulgaris medium is 10 μ g/mL, 20 μ g/mL or 50 μ g/mL; The concentration of described rifampin in described Semen Phaseoli Vulgaris medium is 50 μ g/mL-100 μ g/mL; The concentration of described penicillin in described Semen Phaseoli Vulgaris medium is 50 μ g/mL, 70 μ g/mL or 100 μ g/mL;
Shown in described II, in medium, the concentration of described Difenoconazole in described Semen Phaseoli Vulgaris medium is 10 μ g/mL, 15 μ g/mL or 20 μ g/mL; The concentration of described Prochloraz in described Semen Phaseoli Vulgaris medium is 10 μ g/mL, 20 μ g/mL or 50 μ g/mL; The concentration of described rifampin in described Semen Phaseoli Vulgaris medium is 50 μ g/mL, 70 μ g/mL or 100 μ g/mL; The concentration of described penicillin in described Semen Phaseoli Vulgaris medium is 50 μ g/mL, 70 μ g/mL or 100 μ g/mL.
5. the application of the described medium of claim 3 or 4 in separating Peronophythora Litchii germ (Peronophythora Litchi Chen ex Ko et al).
6. a method of separating Peronophythora Litchii germ (Peronophythora Litchi Chen ex Ko et al), comprise the steps: to obtain the Peronophythora Litchii germ with the described medium culture sample of claim 3 or 4; Described sample is blade or the fruit of the plant of infection Peronophythora Litchii germ.
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