CN1164738C - Insect pestilence germ and method of application for breeding destroy intestinal worms - Google Patents
Insect pestilence germ and method of application for breeding destroy intestinal worms Download PDFInfo
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- CN1164738C CN1164738C CNB031127800A CN03112780A CN1164738C CN 1164738 C CN1164738 C CN 1164738C CN B031127800 A CNB031127800 A CN B031127800A CN 03112780 A CN03112780 A CN 03112780A CN 1164738 C CN1164738 C CN 1164738C
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- xanthomonas campestris
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Abstract
The present invention relates to xanthomonas which can result in various injurious insects of crops to get ill to die and a method of application for breeding destroy intestinal worms. The xanthomonas of the present invention is collected in China Center for Type Culture Collection (CCTCC) in Wuhan on 13, December in 2002, the collected code mark is: CCTCCNO: M 202046, and a Latin scientific name is Xanthomonas. campestris GXW 15-4. By experimental verification, the Xanthomonas. campestris GXW 15-4 can effectively kill various injurious insects of crops, and is safely used. The xanthomonas has no toxic hazard and no pollution for the environment, and can be used as new generational biological insecticide.
Description
Technical field
The present invention relates to a kind of bacterium, the especially a kind of Xanthomonas campestris and disinsection culture using method thereof that can cause the sick death of multiple kinds of crops insect belongs to microbial technology field.
Background technology
Lepidopterous insects is most of farm crop, the insect of greengrocery crop especially.For a long time, suppress and kill pests is that the mankind strive for the important measures that get agricultural profit always.In recent years, the biological control of insect research makes great progress.According to " vegetables pest biological control " book introduction of Golden Shield press publication and distribution in 2000, the bacillary biological pesticide of promoting in China is mainly the B.t series product at present, for example B.tvar7216, B.Tvarhd-1, B.tvar104 etc.Though these insecticidal materials have certain insecticidal effect, because of it mainly causes death insect by the crystal brood cell toxin that polypide digestion thalline sporozoite produces, because crystal brood cell toxin instability, so its action effect is not ideal enough, mainly shows:
1, insecticidal effect is slower, for example to 1-2 green vegetables in age worm under 28 ℃ of temperature condition, 24 hours mortality ratio has only about 75%;
2, insecticidal spectrum is wide inadequately, and is only effective to white butterfly class larva, and to Noctuidae and the anti-heterodyne of Pyralidae larva;
3, often need and the composite use of chemical pesticide,, cause easily and leave residual hazard in the farm crop, be unfavorable for environment protection to improve preventive effect.
Summary of the invention
The objective of the invention is to: provide a kind of can efficiently kill the multiple kinds of crops insect and safe in utilization, to animals and plants toxicological harmless effect, xanthomonas campestris mutation free from environmental pollution, and provide its disinsection culture using method.
Xanthomonas campestris mutation of the present invention is the preservation at the Chinese representative microbial preservation center (CCTCC) in Wuhan on December 13rd, 2002, deposit number is: CCTCC NO:M 202046, Latin formal name used at school are Xanthomonas campestris GXW15-4 (hereinafter referred is XC-GXW15-4).
Its individual morphology is characterized as: Gram-negative, rod-short, the blunt circle in two ends, incubation time are long slightly then shorter, and approximate spherical, the thalline size is 1-1.2 micron * 4.8-5 micron; Motion has flagellum, but the comparatively small amt of flagellum.
Physio-biochemical characteristics comprise glucose, maltose, sucrose, seminose, polychrom, starch, pectinose, semi-lactosi, trehalose, cellobiose and fructose for can utilize multiple kinds of carbohydrate, but can not utilize rhamnosyl;
Can utilize multiple organic acid and organic alcohol, as seminose, saligenin; Can utilize malate, propionic salt, acetate and succinate, but can not utilize ethanol to generate acetate, can not utilize benzoate, oxalate or tartrate, can not utilize ribitol, inositol, galactitol, do not reduce nitrate, can gelatin hydrolysate, connect that to melt the matchmaker negative.
The cultivation using method of xanthomonas campestris mutation of the present invention (XC-GXW15-4) is:
A, with the bacterial strain of separation and purification with test tube LB slant culture, temperature 24～30, illuminance 3000～50001x, 24～36 hours time, pH value 7-7.2;
B, select the high strain excellent of life rate on the LB inclined-plane;
C, with the strain excellent that filters out inoculation enlarged culturing, condition is identical with steps A;
D, dewatered drying, temperature 45～50 makes water content less than 5%, and bacteria containing amount is 100～15,000,000,000 a/gram, and the back of gathering is preserved standby; Preservation period can reach 90 days under 24～28 ℃ of conditions;
When E, use, be sprayed onto corps leaf surface after the microbial inoculum dilution that will preserve.
Find after the dispenser that thalline infects mutually, crop pests is numerous and confused dead in 1～2 day.Its morphological specificity is cephalledema, downward, and belly rots, and overflows weak yellow liquid, and frowziness.Analytical proof, the sporozoite of XC-GXW15-4 can destroy the composition of insect body internal protein, therefore has direct insecticidal action, and is very remarkable to the insect killing effect, and other animal and plant are had no effect, therefore can be used as biotic pesticide of new generation with ideal effect.
The expert thinks that xanthomonas campestris mutation of the present invention is an entomiasis indigenous bacteria unique in the xanthomonas campestris, and its formal name used at school is XC-GXW15-4.124 sick bacteriums of plantation, 5 gene kinds have been named according to Bergey's Mannual (the 8th) pathology of plants man evaluation successively in 1970 and with xanthomonas; Vanterin (1995) has further compared the homology of Xanthomonas 183 member DNA-NDA hybridization and DNA-rRNA hybridization recently, divide into 20 kinds (groups), except four clear and definite kind (x.aibilineansh, x.fragariaee, x.oryzae and x.populi) outside, newly set up 16 kinds again, the following pathogenic mutation of new x.camlpestis kind all is the bacterium monoid that infects Cruciferae.And xanthomonas campestris mutation (XC-GXW15-4) bacterial strain is the entomiasis indigenous bacteria, be that the insect pathogenic strains is at first found and worked out to xanthomonas, XC-GXW15-4 individual morphology, movement characteristic, Gram-reaction, LB cultural characteristic, worm cultural characteristic, biochemical reactions characteristic and to the different prior biological of lepidoptera pest cause a disease effect and unique distinction are worth accelerating the develop and spread utilization in addition.
Embodiment
Embodiment
The xanthomonas campestris mutation of present embodiment can be extracted from December 13rd, 2002 and at preservation, the deposit number of Wuhan China representative microbial preservation center (CCTCC) be: CCTCCNO:M 202046, Latin formal name used at school are the identical separation and purification bacterial classification of Xanthomonas campestris GXW 15-4, cultivate and are used to kill lepidopteran and other insects through following steps:
A, with the bacterial strain of separation and purification with test tube LB slant culture, 24～30 ℃ of temperature, 30～36 hours time, illuminance 3000～50001x, pH value 7-7.2;
B, carry out the lepidopterous larvae mouth and invade pathogenicity rate test, select strain excellent (repetition test 3～5 times);
C, the strain excellent that filters out is inoculated in SN substratum enlarged culturing, condition is identical with steps A; The semi-solid substratum of SN for making with pulverizing behind the bright silkworm chrysalis drying and sterilizing;
D, dewatered drying, 45～50 ℃ of temperature make the microbial inoculum water content less than 5%, and bacteria containing amount is 100～15,000,000,000 a/gram, makes the back and carry out the vacuum preservation under the condition of 15～20 ℃ of temperature, and preservation period can reach more than half a year;
When E, use, will preserve the microbial inoculum dilution, make 0.6 hundred million/milliliter～1.2 hundred million/milliliter of bacteria containing amounts, be sprayed onto on the corps leaf surface.
To the pathogenic mortality ratio test-results of lepidopteran small white section, Noctuidae and Pyralidae 2～3 instar larvaes be: (24-28 ℃)
Cabbage caterpillar Noctuidae larva Pyralidae larva after the dispenser
24 hours 90～100% 90% above 90～95%
36 hours 95～100% 95% above 90～95%
48 hours 95～100% 95% above 90～100%
On October 23rd, 2002 for understanding XC-GXW15-4 to natural enemy kind and number affects, was 0.6 hundred million/ml of XC-GXW15-4 microbial inoculum at cauliflower field spraying concentration, and investigation area is 1m
2/ chi, the result is as follows:
Handle investigation kind control time (head)
24h 48h 72h
GXW15-4 tachinid 2.1 2.2 2.1
Microbial inoculum parastic wasp 2.3 2.6 2.5
0.6 hundred million/ml Chrysopa 0.4 0.5
Ant 4.3 4.6 4.7
Decamethrin parastic wasp 1.1 0.8 1.4
3000 times of liquid tachinids 0.2
Add the acephatemet Chrysopa
1000 times of liquid ants 3.2 2.1 2.4
This explanation XC-GXW15-4 has no adverse effects to natural enemy insect.
This shows, very good to the killing effect of insect.Test shows and has no effect to other animal and plant.Therefore prove that XC-GXW15-4 can effectively kill various pests, and safe in utilization, and is free from environmental pollution to animals and plants toxicological harmless effect, can be used as the biotic pesticide of a new generation.
Claims (6)
1. an xanthomonas campestris mutation (the Latin formal name used at school is Xanthomonascampestris GXW 15-4), Chinese representative microbial preservation center (CCTCC) preservation on December 13rd, 2002 in Wuhan, deposit number is: CCTCC NO:M 202046, Latin formal name used at school are Xanthomonas campestris GXW 15-4.
2. according to the disinsection culture using method of the described xanthomonas campestris mutation of claim 1, the steps include:
A, with the bacterial strain of separation and purification with test tube LB slant culture, 24～30 ℃ of temperature, illuminance 3000～5000lx, 24～36 hours time, pH value 7-7.2;
B, select the high strain excellent of life rate on the LB inclined-plane;
C, the strain excellent that filters out is inoculated in the substratum enlarged culturing, condition is identical with steps A, and described substratum is pulverized after with bright silkworm chrysalis drying and sterilizing and made;
D, dewatered drying, 45～50 ℃ of temperature make water content less than 5%, and bacteria containing amount is 100～15,000,000,000 a/gram, and the back of gathering is preserved standby;
E, be sprayed onto corps leaf surface after will preserving bacterial classification dilution.
3. according to the described xanthomonas campestris mutation of claim 2 disinsection culture using method, it is characterized in that: the enlarged culturing base of described xanthomonas campestris mutation is pulverized after with bright silkworm chrysalis drying and sterilizing and is made semi-solid.
4. according to claim 2 or 3 described xanthomonas campestris mutation disinsection culture using method, it is characterized in that: described xanthomonas campestris mutation is gathered and is pulverized the back and preserve, condition be water content less than 5%, 15～25 ℃ of temperature, intermittent illumination 3000～5000lx, vacuum.
5. according to the described xanthomonas campestris mutation of claim 4 disinsection culture using method, it is characterized in that: described extension rate is 0.6 hundred million/milliliter～1.2 hundred million/milliliter of bacteria containing amounts.
6, according to the application of the described xanthomonas campestris mutation of claim 1 as biotic pesticide.
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| CNB031127800A CN1164738C (en) | 2003-01-28 | 2003-01-28 | Insect pestilence germ and method of application for breeding destroy intestinal worms |
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| CNB031127800A CN1164738C (en) | 2003-01-28 | 2003-01-28 | Insect pestilence germ and method of application for breeding destroy intestinal worms |
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| CN102342299A (en) * | 2011-08-09 | 2012-02-08 | 镇江市润宇生物科技开发有限公司 | Empedobacter brevis pesticide and preparation method thereof |
| CN103333831A (en) * | 2013-07-01 | 2013-10-02 | 镇江市润宇生物科技开发有限公司 | Method for outdoor domestication and purification of empedobacter brevis GXW15-4 |
| CN104059888B (en) * | 2014-06-17 | 2017-04-26 | 镇江市润宇生物科技开发有限公司 | Noctuid granulosis virus, and preparation method and application of preparation containing virus |
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2003
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