CN100467588C - One strain of streptonmyces lydicus and its application in treatment and prevention of plant's disease - Google Patents
One strain of streptonmyces lydicus and its application in treatment and prevention of plant's disease Download PDFInfo
- Publication number
- CN100467588C CN100467588C CNB2006100656209A CN200610065620A CN100467588C CN 100467588 C CN100467588 C CN 100467588C CN B2006100656209 A CNB2006100656209 A CN B2006100656209A CN 200610065620 A CN200610065620 A CN 200610065620A CN 100467588 C CN100467588 C CN 100467588C
- Authority
- CN
- China
- Prior art keywords
- streptomyces lydicus
- cgmcc
- lydicus
- control agent
- streptomyces
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
This invention discloses the application of Streptomyces lydicus A02 CGMCC No.1654. After fermentation, the strain can produce active metabolite having strong antiseptic effects. The metabolite can be used for manufacturing biological agents for resisting plant fungi.
Description
Technical field
The present invention relates to a strain streptomyces lydicus and an application thereof, particularly this strain streptomyces lydicus and the application in biocontrol of plant disease thereof.
Background technology
The Plant diseases that fungi caused accounts for more than 80% of all Plant diseasess, many great diseases are to suffering heavy losses that agriculture production causes, as the gray mold of fruits and vegetables class crop, anthrax, leaf muld of tomato, rice blast, corn northern and southern leaf blight, wheat scab etc.Wherein the gray mold that is caused by Botrytis cinerea (Botrytis cinerea) is the important disease of a kind of worldwide popularity that the vegetable protecting field cultivation generally takes place; the crop species of its harm in recent years constantly increases; the main disease or the primary disease of fruits and vegetables such as tomato, cucumber, eggplant, summer squash, pimento, capsicum, Kidney bean, leek, strawberry, grape have been become at present; often cause fruit rot, loss is serious.The general 20-30% of field production loss that countries such as America and Europe, Australia, Africa, Japan, India cause because of gray mold, the local eliminating vegetable botrytis that China the Yellow River and Huai He River northeast and greenhouse, northwest are concentrated takes place serious, field production loss about 30%, the serious 50-70% that reaches.At present vegetables still do not have the ideal anti-source material to gray mold in producing, and breeding for disease resistance is made slow progress, and control that therefore should disease still depends on chemical agent.But widely apply chemical pesticide for a long time, not only severe contamination people's living environment, destroy the eubiosis, threaten people and animals' health and safety, and cause the drug-fast generation of causal organism, cause the rampant once again and more difficult control of disease.It is reported that in recent years, some medicaments of preventing and treating the main agricultural chemicals benzimidazoles of two classes of gray mold and dicarboximide class have caused the resistance of ash arrhizus bacteria obviously descends its field efficacy.Simultaneously, the pesticide residue of fruit and vegetable food exceed standard and have also constituted the significant obstacle of its foreign exchange earning.And microbial pesticide comes from nature, have that environment compatibility is good, lasting period length, low toxicity, to characteristics such as people and animals and natural enemies securities, its aboundresources simultaneously is especially one of the important environmental protection approach of diseases of garden stuff control of plant, meets the requirement of sustainable agriculture development.Therefore, microbial pesticide has obtained developing rapidly in the world in recent years.
Streptomyces lydicus (Streptomyces lydicus DeBoer et al.) is to practise the saprophytic microbe occupy soil, existing years of researches history, and a kind of as streptomycete, this bacterium can produce multiple antibacterial substance.Its famous representative strain is S.lydicus NRRL2433, this bacterial strain is studied and is widely used at field of medicaments, can produce to have wide spectrum antibacterium and the lydimycin (Lydimycin) of mycobacterium effect and the streptolydigin (Streptolydigin) of resisting gram-positive bacterium and mycobacterium.Using wider bacterial strain on control of plant disease is S.lydicus WYEC 108, this bacterial strain initial separation is from British agriculture soil, it can grow the tip of a root position plant surely, mainly act on the pathogenic bacteria of soil-borne disease in superparasitic mode, comprise pythium spp (Pythium), rhizoctonia (Rhizoctonia), sickle-like bacteria (Fusarium,), Verticillium (Verticillium), top softgel shell (Gaeumannomyces) etc. belong to the kind of fungi, also can secrete the cell walls that chitinase (chitinase) destroys fungi.This bacterial strain has been developed the biological control that active bacteria formulation is used for diseases such as plant root rot, seed rot abroad.
Summary of the invention
The purpose of this invention is to provide the new streptomyces lydicus of a strain, this bacterial strain back by fermentation produces the active metabolite with strong bacteriostatic action, this active metabolite can be used for preparing the biological prevention and control agent of broad spectrum plant fungal disease resistance, is used for the biological control of plant gas borne fungus diseases.
Streptomyces lydicus provided by the present invention, be streptomyces lydicus (Streptomyces lydicus) A02, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 03 16th, 2006, preservation registration number is CGMCC No.1654.
Streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 separates obtaining from the Natural Secondary Forests soil of outer suburbs, Beijing.Concrete grammar is as follows: gather soil sample from outer suburbs, Beijing Natural Secondary Forests ground, get in the 10g adding and adorn in the triangular flask of 100ml sterilized water and granulated glass sphere, the 20min that vibrates on 100 commentaries on classics/min shaking tables gets 0.5ml and adds in the 4.5ml sterilized water, dilutes 10 successively again
-2, 10
-3, 10
-4Doubly, get above soil supension 0.1ml evenly coating on the Gause I culture medium flat plate respectively, blow in the Bechtop to slightly doing; 3 repetitions of every concentration place 28 ℃ of thermostat containers to cultivate 5-10 days, and the single bacterium colony of picking actinomycetes dilutes the line separation and purification.With pathogenic fungies such as ash arrhizus bacterias is the target bacterium, utilizes dull and stereotyped face-off culture method to carry out antimicrobial primary dcreening operation short of money; The bacterial strain that antagonism is arranged that primary dcreening operation is obtained carries out the shake flask fermentation cultivation, and fermented liquid obtains aseptic ferment filtrate through the aseptic filtering with microporous membrane degerming of 0.45 μ m; Further this aseptic ferment filtrate of test is to the restraining effect and the greenhouse protection effect of pathogenic bacteria mycelial growth, spore germination, and finishing screen is selected the good biocontrol strain streptomyces lydicus of proterties (Streptomyces lydicus) A02 CGMCC No.1654.
The result of morphological feature, cultural characters, physio-biochemical characteristics and the 16SrDNA sequential analysis of comprehensive streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 is accredited as streptomyces lydicus with it.Concrete qualification result is as follows:
(1) morphological specificity of thalline
Gram-positive; At GYM agar, JCM42
#Growth is after 7 days on the substratum such as agar, oatmeal agar, and substrate mycelium physically well develops, and no tabula does not rupture; The aerial hyphae well-grown, multi-branched; Fibrillae of spores is gentle bent or crooked, the spore ellipse.
(2) cultural characters
Cultural characters on 6 kinds of solid mediums is as shown in table 1.
The cultural characters of table 1 streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654
| Substratum | Aerial hyphae | Substrate mycelium | But lysochrome |
| Gao Shi synthesizes No. 1 agar ISP4 agar GYM agar Bennett ' s agar JCM42 #Substratum oatmeal agar | The light gray lime is yellow greyish white greyish white to the greyish white ecru of sallow | The brown yellowish-brown brown ash of the yellowish-brown orange of little Huang | It is pale yellow yellow not have no shallow palm fibre |
(3) physio-biochemical characteristics
The physio-biochemical characteristics of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 are as shown in table 2.
The physio-biochemical characteristics of table 2 streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654
| Characteristic | The result | Characteristic | The result |
| I. growth and 45 ℃ of Mierocrystalline cellulose starch-splittings of other 3%NaCl 5%NaCl 7%NaCl 10%NaCl hydrolysis Vitamin C2 degraded gelatin liquefaction Citrate trianion produces sour II. utilization of carbon source seminose glucosylmannitol | + + W - - - - + + - W + + | II. utilization of carbon source sorbyl alcohol sorbose synanthrin fructose lactose semi-lactosi pectinose ribose rhamnosyl raffinose sucrose wood sugar inositol Alpha-Methyl-D-glucoside malonate | + - - W + + - - - + + - + W - |
Annotate: the weak positive findings of " W " expression; "+" expression positive findings; "-" expression negative findings.
4.16SrDNA sequential analysis
The part 16SrDNA sequence of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 is shown in sequence in the sequence table 1.Show that with the result relatively of correlated series Blast among the GenBank it belongs to streptomyces; It is very high that the relevant bacterial strain with the streptomyces lydicus of delivering at present of 16SrDNA sequence of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 is compared similarity, is 99.9%.
Streptomyces lydicus of the present invention (Streptomyces lydicus) A02 CGMCC No.1654 can produce great deal of bioactive substances in substratum.Dull and stereotyped face-off culture experiment shows that this active metabolite is to ash arrhizus bacteria (Botrytis cinerea), tomato early blight bacterium (Alternayia solani), fusarium graminearum (Fusarium graminearium), leaf muld of tomato bacterium (Fulvia fulva), celery septoria disease bacterium (septoria apiicola), big purple blotch of onion bacterium (Alternaria poprri), rice blast fungus (Pyricularia oryzae), the pathogenic bacteria of Exserohilum turcicum various plants fungoid air infection diseasess such as (Exserohilum turcicum) has the obvious suppression effect; But the experiment of measuring inhibition zone by dull and stereotyped punch method and filter paper method shows that it does not have bacteriostatic activity to cucumber bacterial angular leaf spot bacterium (Pseudomonas syringae pv.lachrymans), pepper scab fungus (Xanthomonas campestris pv.vesicatoria), Agrobacterium tumefaciens (Agrobacterium tumefaciens), subtilis (Bacillus subtilis), intestinal bacteria bacteriums such as (Escherichia coli); Directly face-off cultivate to be observed and sclerotium superparasitism evidence in culture dish, and this bacterial strain is not had a bacterium parasitics.As seen its antimicrobial spectrum is obviously different with aforementioned known bacterial strain S.lydicus NRRL2433, and its main bacteriostatic action mode is different with WYEC 108 then.
Streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654, be inoculated in seed culture medium, 28 ℃, behind the 200rpm/min shake-flask culture 24h-30h, grain weight inoculation fermentation substratum by 5% (volume ratio), liquid amount is a standard with 1/5 of triangular flask volume, under 31 ℃ of conditions, rotation radius with 13mm, the rotating speed shake flask fermentation 96h of 240rpm/min, can make this bacterial strain produce the bacteriostatic activity meta-bolites of high density in fermented liquid, the bacteria-free filtrate of its fermented liquid can reach more than the 40mm the antibacterial circle diameter of ash arrhizus bacteria, and the greenhouse pot culture protection effect is 82%-90%.
Another object of the present invention provides a kind of biological prevention and control agent of plant fungal disease resistance.
The biological prevention and control agent of plant fungal disease resistance provided by the present invention is the meta-bolites that fermentation streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 obtains.
Described meta-bolites can obtain from the ferment filtrate of removing streptomyces lydicus (Streptomyces lydicus) A02 CGMCCNo.1654 thalline.
The pH of described streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 fermention medium is 7-8, be made into by following material: the 15g soybean grain, the 5g peptone, 2.5g ammonium sulfate, 10g sucrose, 10g starch, 0.25g sal epsom, 0.2g potassium primary phosphate, 5g sodium-chlor, 1g lime carbonate adds water and is settled to 1000ml.Wherein, described 15g soybean grain is that the 15g soybean grain is added water boil 0.5-1h, gets filtrate.
The leavening temperature of described streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 can be 28-31 ℃.
Air flow in the described fermentation can by liquid amount be shake bottle long-pending 1/5, the condition control of 13mm rotation radius 240rpm/min vibration; Fermentation time can be 90-100 hour.
The present invention also provides the preparation method of the biological prevention and control agent of above-mentioned plant fungal disease resistance.
The preparation method of the biological prevention and control agent of plant fungal disease resistance provided by the present invention, be fermentation streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654, from its fermented liquid, separate the biological prevention and control agent that the meta-bolites that obtains is plant fungal disease resistance.
The pH of described streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 fermention medium is 7-8, be made into by following material: the 15g soybean grain, the 5g peptone, 2.5g ammonium sulfate, 10g sucrose, 10g starch, 0.25g sal epsom, 0.2g potassium primary phosphate, 5g sodium-chlor, 1g lime carbonate adds water and is settled to 1000ml.Wherein, described 15g soybean grain is that the 15g soybean grain is added water boil 0.5-1h, gets filtrate.
The leavening temperature of described streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 is 28-31 ℃; Air flow in the described fermentation can by liquid amount be shake bottle long-pending 1/5, the condition control of 13mm rotation radius 240rpm/min shaking culture; Fermentation time is 90-100 hour.
The aseptic ferment filtrate (biological prevention and control agent of plant fungal disease resistance) that experiment showed, streptomyces lydicus of the present invention (Streptomyces lydicus) A02 CGMCC No.1654 all has good preventive effect to graw mold of tomato and pimento gray mold; Its 2 times of diluents reach 89.34% and 90.04% respectively to the average preventive effect of the two, and variance analysis and average The result of multiple comparisons show that its effect is significantly higher than the good medication 50% grey health suspension agent of gray mold control in the present production; Its 5 times of diluents reach 83.63% and 82.74% respectively to the average preventive effect of the two, do not have significant difference with the preventive effect of grey health.Therefore, streptomyces lydicus (Streptomyces lydicus) A02 CGMCCNo.1654 and in the control of fruits and vegetables gray mold, good prospects for application is arranged with the biological prevention and control agent of plant fungal disease resistance of its preparation.
The aseptic ferment filtrate (biological prevention and control agent of plant fungal disease resistance) of streptomyces lydicus of the present invention (Streptomyces lydicus) A02 CGMCC No.1654 is to the sprouting of tomato and pimento seed and take root obvious facilitation is arranged, and other is not had restraining effect for the seed germination that studies thing.
Description of drawings
Fig. 1 is the flat board face-off culture experiment of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 and botrytis cinerea pers
Fig. 2 is the dull and stereotyped photo that the aseptic ferment filtrate of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 suppresses the botrytis cinerea mycelial growth
Fig. 3 is the dull and stereotyped photo of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 to the inhibition zone of botrytis cinerea generation
Fig. 4 is the restraining effect photo of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 to the botrytis cinerea conidia germination
Fig. 5 is the greenhouse preventive effect photo of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 ferment filtrate to graw mold of tomato
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Realize that substratum tool of the present invention is as described below:
1, streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 plate isolation and inclined-plane are preserved and are adopted Gause I substratum: K
2HPO
40.5g, NaCl 0.5g, KNO
31.0g, FeSO
47H
2O0.01g, MgSO
47H
2O 0.5g, Zulkovsky starch 20g, agar 20g, water 1000ml; PH7.2-7.4.
2, dull and stereotyped face-off is cultivated and is adopted the PDA substratum: potato 200g, sucrose 10-20g, agar 17-20g, water 1000ml; The pH nature.
3, the streptomyces lydicus that obtains through optimization of orthogonal test (Streptomyces lydicus) A02 CGMCCNo.1654 fermention medium: 15g soybean grain (adding distil water boils 0.5-1h, gets filtrate), 5g peptone, 2.5g ammonium sulfate, 10g sucrose, 10g starch, 0.25g sal epsom, 0.2g potassium primary phosphate, 5g sodium-chlor is made into the aqueous solution, behind the accent pH7-8, add 1g lime carbonate, add water and be settled to 1000ml.0.10342Mpa sterilization 20min.
4, seed culture medium: the 10g sucrose in the fermentative medium formula is replaced 20g glucose, other components unchanged.
The biological prevention and control agent specimen preparation of embodiment 1, streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 spawn culture and plant fungal disease resistance
Streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 is inoculated on the above-mentioned Gause I slant medium, cultivated 7-10 days for 28 ℃, treat that it produces the spore of capacity, scrape with aseptic platinum loop and to get its spore 2-3 rings and be inoculated in the above-mentioned seed culture medium of 50ml in the 250ml triangular flask, put on the temperature controllable shaking table, under 28 ℃ of conditions, 200rpm/min (rotation radius 13mm) constant-temperature shaking culture 24h-30h; Under aseptic condition, its branch is connected to then in the above-mentioned fermention medium in 10 500ml triangular flasks (every bottled liquid measure is 100ml); The postvaccinal bottle that shakes is cultivated 96h with the speed oscillation of 240rpm/min (rotation radius 13mm) under 31 ℃ of conditions; This moment, bacterial strain produced the bacteriostatic activity meta-bolites of high density in fermented liquid.
Fermented liquid 5000rpm/min centrifugal 10-15min sedimentation mycelium and solid substance with above step acquisition, supernatant liquor is with the aseptic filtering with microporous membrane of 0.45 μ m, collect filtrate (not having streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 thalline in this filtrate), the aseptic ferment filtrate of this streptomyces lydicus (Streptomyceslydicus) A02 CGMCC No.1654 is the biological prevention and control agent sample of plant fungal disease resistance, and it is standby to put 4 ℃ of storages.
The bacteriostatic activity test of embodiment 2, streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 and meta-bolites thereof
Substratum for examination has in the present embodiment: Gause I substratum, PDA substratum, seed culture medium and fermention medium, and it is formed all as mentioned above; For examination target pathogenic bacteria is 3 bacterial strains of Botrytis cinerea bacterium (Botrytis cinereaPers.), i.e. botrytis cinerea pers, botrytis cinerea and pimento ash arrhizus bacteria.
1. dull and stereotyped face-off culture method is adopted in the test of the bacteriostatic activity of flat-plate bacterial colony, earlier the spore of streptomyces lydicus on the solid inclined-plane (Streptomyces lydicus) A02 CGMCC No.1654 is made bacteria suspension with sterilized water, getting 200-500 μ l bacteria suspension is evenly coated on the Gause I solid plate of diameter 90mm, cultivate 2-4d, make it cover with flat board for 28 ℃; Aseptic punch tool with diameter 7mm is made the bacterium cake, then at dull and stereotyped side joint kind streptomyces lydicus (Streptomyces lydicus) the A02 CGMCC No.1654 bacterium cake of previously prepd PDA, the control strain bacterium cake of the no antagonism of opposite side symmetric position inoculation, cultivate after 2 days, utilize the PDA flat board to make target pathogenic bacteria bacterium cake for 28 ℃ with same method; At the above-mentioned dull and stereotyped central authorities of the PDA inoculation target pathogenic bacteria bacterium cake of having inoculated streptomyces lydicus (Streptomyceslydicus) A02 CGMCC No.1654 and control strain, cultivate down, observe the antagonism reaction for 28 ℃.Three repetitions of every processing.
The result that face-off is cultivated as shown in Figure 1, inoculation target pathogenic bacteria produces tangible antibacterial band between streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 and target bacterial strain botrytis cinerea pers, botrytis cinerea and pimento ash arrhizus bacteria bacterium colony after 4 days, its width is respectively 20.9mm, 21.2mm and 22.4mm, and antibacterial bandwidth not in time passing and change; Between control strain and target pathogenic bacteria bacterium colony, then do not have antibacterial band and produce (Fig. 1).Among Fig. 1, A02 is streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654, and yellow ash is a botrytis cinerea pers, and CK is the control strain of no antagonism.
2. bacterial strain meta-bolites bacteriostatic activity is measured
The method of employing embodiment 1 prepares the confession examination fungistat (aseptic ferment filtrate) of streptomyces lydicus (Streptomyces lydicus) A02 CGMCCNo.1654, measures following index then respectively:
(1) mycelial growth rate suppresses to measure: streptomyces lydicus (Streptomyces lydicus) the A02 CGMCC No.1654 for preparing is not had fermented liquid mix with the PDA substratum, be prepared into respectively and contain 5,10,20,100, the PDA flat board of 200 times of dilution fermented liquids is contrast with the PDA flat board that contains corresponding extension rate fermention medium; Dull and stereotyped central inoculating tomato ash arrhizus bacteria bacterium cake is cultivated down for 28 ℃, respectively the 1st, 2, calculates mycelial growth inhibition rate with right-angled intersection method measurement colony diameter in 3,4,5 days.Three repetitions of every processing.
Experimental result is as shown in table 3, the aseptic ferment filtrate of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 different concns all has significant inhibitory effect to the botrytis cinerea mycelial growth, its inhibiting rate reduces along with the increase of ferment filtrate extension rate, descends to some extent along with time lengthening.Handling after 120h, its 5 times and 10 times of diluents still reach 100% to the inhibiting rate of pathogenic bacteria mycelial growth, promptly can suppress the growth (Fig. 2) of botrytis cinerea fully.Among Fig. 2, CK is not for inoculating the fermention medium of streptomyces lydicus (Streptomyceslydicus) A02 CGMCC No.1654, and 5 times, 10 times, 20 times, 100 times, 200 times are respectively the extension rate that streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 does not have fermented liquid.
The aseptic ferment filtrate of table 3 streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 is to the inhibition of botrytis cinerea mycelial growth rate
Annotate: CK is the dull and stereotyped contrast of PDA that contains corresponding extension rate fermention medium.
(2) inhibition zone is measured: adopt dull and stereotyped punch method.Scrape to get on the PDA flat board and cultivate the botrytis cinerea that produces and the conidium of pimento ash arrhizus bacteria, make bacteria suspension, be applied to equably on the freshly prepd PDA flat board, put in the Bechtop and dry up with sterilized water; Aseptic punch tool symmetric position punching around flat board with diameter 7mm, injecting the aseptic ferment filtrate 100 μ l of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 in every then hole, is contrast with the fermention medium of not inoculating streptomyces lydicus (Streptomyces lydicus) A02 CGMCCNo.1654; Cultivate 3d down for 28 ℃, the right-angled intersection method is measured antibacterial circle diameter.Three repetitions of every processing.
Experimental result shows that the aseptic ferment filtrate of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 can all produce tangible inhibition zone to botrytis cinerea and pimento ash arrhizus bacteria on the PDA flat board, and its three multiple average diameter of inhibition zone are respectively 41.0mm and 40.4mm (Fig. 3).Among Fig. 3, A, B, C are the aseptic ferment filtrate of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654; D is contrast.
(3) the spore germination inhibiting rate is measured: scrape the botrytis cinerea conidium that produces on the plate of making even, be made into the bacteria suspension of every visual field 50-100 spore under 10 * 10 power microscopes with fermention medium, be diluted to 5 times with its aseptic ferment filtrate then with streptomyces lydicus A02 (CGMCC No.1654), 10 times, 20 times, 100 times, 5 various concentrations over control treatment such as 200 times of grades, with the fermention medium of not inoculating streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654 is contrast, under 28 ℃, leave standstill cultivation with aseptic depression slide, respectively at handling back 4h, 8h, 12h, 24h microscopy spore germination situation, half that surpasses the conidium diameter with germ tube length is as the sprouting standard, and statistics is sprouted quantity and calculated and sprout inhibiting rate; Three repetitions of every processing.
The result shows the restraining effect very remarkable (as table 4) of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 to the botrytis cinerea conidia germination; Its aseptic ferment filtrate extension rate≤almost can suppress the germ spore germination fully at 20 o'clock; Extension rate during less than 100 times, though can not suppress spore germination fully, can significantly reduce its germination rate and germ tube elongation speed greater than 20 times; The germ tube top of 100 times of diluents after to spore germination has tangible teratogenesis restraining effect, makes it no longer continue elongation at least in 24h, loses the ability (Fig. 4) that infects the host.Among Fig. 4,1 is 100 times of diluents processing back 24h of the aseptic ferment filtrate of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654; 2 is 24h after the control treatment.
The aseptic ferment filtrate of table 4 S.lydicus A02 is to the inhibition of botrytis cinerea conidia germination
The greenhouse diseases prevention experiment of embodiment 3, streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654
For trying the target pathogenic bacteria: botrytis cinerea and pimento ash arrhizus bacteria
For studying the article kind; Middle No. 6 tomatoes of vegetables, eggplant door pimento
Contrast medicament for examination: 50% grey health suspension agent (agriculture chemical registration Ls20041062, radically reform and protect bio tech ltd in the Inner Mongol)
The aseptic ferment filtrate for preparing streptomyces lydicus (Streptomyces lydicus) A02 CGMCCNo.1654 according to the method for embodiment 1.Botrytis cinerea and pimento ash arrhizus bacteria on the PDA flat board 22 ℃ cultivated 7-10 days, treat that it grows a large amount of conidiums after, wash spore with sterilized water, the bacteria suspension that is mixed with every visual field 30-50 spore under 10 * 10 times of mirrors is standby.
4 processing are established in experiment: 5 times of diluents of aseptic ferment filtrate of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 and 2 times of diluents (A02 ferment filtrate (5 *) and A02 ferment filtrate (2 *)); 1000 times of diluents of 50% grey health suspension agent (50% grey health (1000 *)); The clear water contrast.Each handles 15 young plants, three repetitions; Each handles random alignment.
Grow to the inoculation in flowering period of buddingging at tomato and pimento seedling, evenly spray earlier and respectively handle medicament and clear water contrast, with the blade face degree of being that drips; After treating that the blade face is dried slightly, the spore suspension of spray inoculation botrytis cinerea and pimento ash arrhizus bacteria again.The inoculation back hides with plastics film, and with the humidifier humidification, preserves moisture 48 hours under 22 ℃ of conditions; After removing covering, often water spray does not make overdrying.Investigation in 7-10 days incidence in inoculation back calculates sickness rate, disease index and prevention effect, and utilizes statistical analysis software SPSS11.0, selects the Duncan method that experimental result is carried out variance analysis and multiple comparisons.
State of an illness investigation is that unit generally investigates with the blade, and the disease grade scale is as follows:
0 grade: no scab;
1 grade: single blade has 3 of scabs;
3 grades: single blade has scab 4-6;
5 grades: single blade has scab 7-10;
7 grades: single blade has scab 11-20, and part is intensive in flakes;
9 grades: single blade has the intensive leaf area that accounts for of scab more than 25%;
Disease index=(∑ (the sick numbers of sheets at different levels * relative level numerical value)/(investigating total number of sheets * 9)) * 100
Prevention effect (%)=((contrast disease index-processing disease index)/contrast disease index) * 100
Experimental data and statistic analysis result such as table 5 are to table 8 and Fig. 5.Among Fig. 5,1 is 50% grey health (1000 *); 2 is A02 ferment filtrate (2 *); 3 is A02 ferment filtrate (5 *): 4 are the clear water contrast.
Table 5. streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 ferment filtrate is to the greenhouse prevention effect of graw mold of tomato
Table 6 Duncan method his-and-hers watches 5 carry out the homogeneous subclass result of average multiple comparisons
Table 7. streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 ferment filtrate is to the greenhouse prevention effect of pimento gray mold
Table 8 Duncan method his-and-hers watches 7 carry out the homogeneous subclass result of average multiple comparisons
Table 5-8 shows that the ferment filtrate of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 all has good preventive effect to graw mold of tomato and pimento gray mold; Its 2 times of diluents reach 89.34% and 90.04% respectively to the average preventive effect of the two, and variance analysis and average The result of multiple comparisons show that its effect is significantly higher than the good medication 50% grey health suspension agent of gray mold control in the present production; Its 5 times of diluents reach 83.63% and 82.74% respectively to the average preventive effect of the two, do not have significant difference with the preventive effect of grey health.Therefore, streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 and in the control of fruits and vegetables gray mold, good prospects for application is arranged with the biological prevention and control agent of plant fungal disease resistance of its preparation.
Use according to the aseptic ferment filtrate of streptomyces lydicus (Streptomyces lydicus) A02 CGMCCNo.1654 of the method for embodiment 1 preparation and handle 8 kind of plant seeds such as tomato, pimento, cucumber, Chinese cabbage, eggplant, wild cabbage, wheat, corn, the result shows that this biological prevention and control agent to the sprouting of tomato and pimento seed with take root obvious facilitation is arranged, does not have restraining effect to other for the seed germination that studies thing.
The mensuration of embodiment 4, bacterial strain streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 antimicrobial spectrum
Adopt the flat board face-off culture method among the embodiment 2, on the PDA flat board, detect streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 to 17 restraining effect, and utilize SPSS software to carry out variance analysis and multiple comparisons for examination target plant pathogenic fungi bacterial strain.The result is as shown in table 9, and streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 all has the obvious suppression effect to the whole pathogenic bacterias for examination; These pathogenic bacterias totally 9 belong to 12 kinds, almost in each order of Deuteromycotina two outlines distribution are arranged all; They have certain difference to the susceptibility of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654, and the result of Duncan method multiple comparisons is divided into 7 homogeneous subclass with it; The susceptibility of total 6 bacterial strains seeing ash arrhizus bacteria and tomato early blight bacterium, fusarium graminearum, melon anthrax bacteria is higher.
Only be the minority representative species of the plant pathogenic fungi selected for use at random for the examination target in the present embodiment, and they have susceptibility to streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 bar none; As seen the antimicrobial spectrum of bacterial strain streptomyces lydicus of the present invention (Streptomyces lydicus) A02 CGMCC No.1654 can not be so limited, and it should be a wide spectrum to imperfect fungi at least.
Table 9 streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 is to the antagonistic effect for the examination pathogenic bacteria
| Sequence number | For the examination pathogenic bacteria | Antibacterial band average (mm) | The significance of difference |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 | ( Alternaria alternata ) ( Alternaria poprri ) ( Alternaria solani ) ( Ascochyta lycopersici ) ( Botrytis cinerea ) ( Botrytis cinerea ) ( Botrytis cinerea ) ( Botrytis cinerea ) ( Botrytis cinerea ) ( Botrytis cinerea ) ( Colletotrichum orbicular ) ( Colletotrichum capsici ) ( Exserohilum turcicum ) ( Fulvia fulva ) ( Fusarium graminearium ) ( Phomopsis vexans ) ( Pyricularia oryzae ) | 16.6 15.7 22.4 17.5 20.9 21.2 22.4 21.5 19.5 20.8 18.7 17.9 17.3 16.8 21.7 17.4 16.6 | fg g a ef b b a ab c b cd de ef f ab ef fg |
Annotate: alphabetical a-g represents 7 homogeneous subclass respectively, and all each bacterial strains that indicates same letter are returned in same subclass, and its susceptibility to bacterial strain streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 does not have significant difference
Sequence table
<160>1
<210>1
<211>1361
<212>DNA
＜213〉streptomyces lydicus (Streptomyces lydicus)
<400>1
Claims (10)
1, streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654.
2, the application of streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 in the biological prevention and control agent of preparation plant fungal disease resistance.
3, the biological prevention and control agent of plant fungal disease resistance is the meta-bolites that fermentation streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 obtains.
4, biological prevention and control agent according to claim 3 is characterized in that: described meta-bolites obtains from the ferment filtrate of removing streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 thalline.
5, biological prevention and control agent according to claim 4 is characterized in that: the pH of the fermention medium of described streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 is 7-8, is made into by following material: the 15g soybean grain, the 5g peptone, 2.5g ammonium sulfate, 10g sucrose, 10g starch, 0.25g sal epsom, 0.2g potassium primary phosphate, 5g sodium-chlor, 1g lime carbonate, add water and be settled to 1000ml, wherein soybean grain is that adding distil water boils 0.5-1h, gets filtrate; The leavening temperature of described streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654 is 28-31 ℃; Fermentation time is 90-100 hour.
6, biological prevention and control agent according to claim 5 is characterized in that: described fungal diseases of plants is a gray mold.
7, biological prevention and control agent according to claim 6 is characterized in that: described fungal diseases of plants is graw mold of tomato and/or pimento gray mold.
8, a kind of method for preparing the biological prevention and control agent of arbitrary described plant fungal disease resistance among the claim 3-7, be fermentation streptomyces lydicus (Streptomyces lydicus) A02 CGMCC No.1654, from its fermented liquid, separate the biological prevention and control agent that the meta-bolites that obtains is plant fungal disease resistance.
9, method according to claim 8 is characterized in that: the pH of the fermention medium of described streptomyces lydicus (Streptomyceslydicus) A02 CGMCC No.1654 is 7-8, is made into by following material: the 15g soybean grain, the 5g peptone, 2.5g ammonium sulfate, 10g sucrose, 10g starch, 0.25g sal epsom, 0.2g potassium primary phosphate, 5g sodium-chlor, 1g lime carbonate, add water and be settled to 1000ml, wherein soybean grain is that adding distil water boils 0.5-1h, gets filtrate.
10, method according to claim 9 is characterized in that: the leavening temperature of described streptomyces lydicus (Streptomyceslydicus) A02 CGMCC No.1654 is 28-31 ℃; Air flow in the described fermentation by liquid amount be shake bottle long-pending 1/5, the condition control of 13mm rotation radius 240rpm/min shaking culture, fermentation time is 90-100 hour.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100656209A CN100467588C (en) | 2006-03-21 | 2006-03-21 | One strain of streptonmyces lydicus and its application in treatment and prevention of plant's disease |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100656209A CN100467588C (en) | 2006-03-21 | 2006-03-21 | One strain of streptonmyces lydicus and its application in treatment and prevention of plant's disease |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1834230A CN1834230A (en) | 2006-09-20 |
| CN100467588C true CN100467588C (en) | 2009-03-11 |
Family
ID=37002127
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2006100656209A Active CN100467588C (en) | 2006-03-21 | 2006-03-21 | One strain of streptonmyces lydicus and its application in treatment and prevention of plant's disease |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100467588C (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100590194C (en) * | 2007-09-29 | 2010-02-17 | 北京市农林科学院 | Streptomyces lydicus producing natamycin and uses thereof |
| CN101805717B (en) * | 2010-04-12 | 2012-03-14 | 河北省农林科学院植物保护研究所 | Method for efficiently and directionally screening biocontrol bacteria in soil-borne diseases of crops |
| CN102835423B (en) * | 2012-08-01 | 2014-04-16 | 淮海工学院 | Streptomyces mediolani ZW-1 bacterial strain, and bacteriostatic application of fermentation broth thereof |
| CN102965389B (en) * | 2012-11-23 | 2014-03-12 | 北京市农林科学院 | Recombinant streptomyces lydicus with cellulase activity and construction method and application thereof |
| CN103045516B (en) * | 2012-12-27 | 2014-07-09 | 北京市农林科学院 | Novel strain for producing natamycin and application thereof |
| CN106834193B (en) * | 2017-03-28 | 2020-08-04 | 陕西枫丹百丽生物科技有限公司 | Biocontrol bacterium for preventing and treating fungal diseases of fruits and vegetables |
| CN109837228B (en) * | 2019-04-03 | 2020-03-03 | 南京工业大学 | Streptomyces lydicus and application thereof in prevention and treatment of fire blight |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5286745A (en) * | 1991-09-09 | 1994-02-15 | American Cyanamid Company | Antibiotics LL-E19020 Epsilon and LL-E19020 Epsilon1 |
-
2006
- 2006-03-21 CN CNB2006100656209A patent/CN100467588C/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5286745A (en) * | 1991-09-09 | 1994-02-15 | American Cyanamid Company | Antibiotics LL-E19020 Epsilon and LL-E19020 Epsilon1 |
Non-Patent Citations (1)
| Title |
|---|
| 放线菌III－61和A－21对蔬菜枯萎病和灰霉病的控制作用. 潘争艳.华北农学报，第20卷第4期. 2005 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1834230A (en) | 2006-09-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100334201C (en) | Bacillus subtilis and its uses | |
| CN103087951B (en) | Bacillus amyloliquefaciens and application thereof | |
| Shan et al. | Biocontrol of rice blast by the phenaminomethylacetic acid producer of Bacillus methylotrophicus strain BC79 | |
| CN102433282B (en) | Bacillus subtilis NB12, as well as culture method and application thereof | |
| CN104762223B (en) | A kind of bacillus amyloliquefaciens BA-KA3 and its application | |
| CN107083349B (en) | The white black streptomycete of one plant of disease prevention growth-promoting and its preparation and application of metabolite | |
| CN102433281B (en) | Streptomyces katrae NB20, as well as culture method and application thereof | |
| CN105886428A (en) | Streptomyces albidoflavus and applications thereof in microbial fertilizers | |
| CN100467588C (en) | One strain of streptonmyces lydicus and its application in treatment and prevention of plant's disease | |
| CN101531971B (en) | Bacillus subtilis and application thereof in prevention and treatment of continuous cropping obstacle of greenhouse vegetable | |
| CN101148650A (en) | Bacillus subtilis, bacterium agent and application thereof | |
| CN103756931A (en) | Paenibacillus kribbensis and its application | |
| CN101698827B (en) | Erythrochromogenes and use thereof in biological control of diseases | |
| CN104673705B (en) | One plant prevent and treat wheat scab bacillus amyloliquefaciens and its application | |
| CN101781626A (en) | Streptomyces avermitilis with antagonism to pyriculariagrisea and preparation method thereof | |
| CN103087960A (en) | Bacillus amyloliquefaciens FQS38 and application thereof | |
| CN106350464A (en) | Microbial agent for preventing and treating cumquat huanglongbing and preparation method thereof | |
| CN100512651C (en) | Biocontrol fungus for preventing and controlling plant mycosis and its preparation process | |
| CN103667099B (en) | A kind of subtilis for preventing and treating crop fusarium disease and application thereof | |
| CN101518264B (en) | Microbial preparation for preventing and controlling plant silborne fungal diseases, and application thereof | |
| KR101175532B1 (en) | Novel burkholderia sp., and media for mass culture of burkholderia for increasing antifungal effect and method for mass production thereof using the same | |
| US20240032545A1 (en) | Streptomyces ardesiacus, microbial biological control agent (mbca) and use | |
| CN107629985A (en) | One plant of endophytic bacterium for having antagonism to plant pathogenic fungi | |
| CN107502570A (en) | One plant of Biocontrol Bacillus subtilis BJ 1 and its application | |
| CN105087428A (en) | Pseudomonas aeruginosa and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: ZHONGNONG LVKANG (BEIJING) BIOTECHNOLOGY CO., LTD. Free format text: FORMER OWNER: BEIJING AGRICULTURE AND FORESTRY ACADEMY OF SCIENCE Effective date: 20140611 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 100089 HAIDIAN, BEIJING TO: 102101 YANQING, BEIJING |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20140611 Address after: 102101, No. 8, East Ring Road, Badaling Economic Development Zone, Yanqing, Beijing Patentee after: The middle peasants lvkang Biotechnology Co. Ltd. (Beijing) Address before: 100089 environmental protection and Environmental Protection Institute of Beijing Academy of agriculture and Forestry Sciences, Haidian District City, Beijing Patentee before: Beijing Academy of Agriculture and Forestry Sciences |