CN102925387B - Bacillus simplex for inducing soybean to generate soybean cyst nematode resistance and application - Google Patents
Bacillus simplex for inducing soybean to generate soybean cyst nematode resistance and application Download PDFInfo
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- CN102925387B CN102925387B CN201210414207.4A CN201210414207A CN102925387B CN 102925387 B CN102925387 B CN 102925387B CN 201210414207 A CN201210414207 A CN 201210414207A CN 102925387 B CN102925387 B CN 102925387B
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Abstract
The invention relates to a method for culturing a Bacillus simplex Sneb545 for inducing soybean to generate soybean cyst nematode resistance after soybean seed treatment and an application of the Bacillus simplex Sneb545, and belong to the technical field of microbial pesticides. The bacterial strain Sneb545 related in the invention is preserved in the China general microbiological culture collection center in October 11th, 2012, and the preservation number is CGMCC No.6650. After the strain is subjected to fermentation cultivation through conventional liquid, the metabolic products of the strain can be used to develop a seed treating agent for treating soybean seeds, and the treated soybean seeds can induce the soybeans to generate the soybean cyst nematode resistance. The soybean seeds which is treated through the metabolic products of the Bacillus simplex Sneb545 can induce the soybeans to generate obvious resistance to the first generation of soybean cyst nematode during the seedling stage, the Bacillus simplex Sneb545 has an obvious function of inhibiting the cytocyst of the soybean cyst nematode from forming and is a potential biocontrol bacterium, and a new method is provided for preventing the soybean cyst nematode.
Description
Technical field
The invention belongs to microbial pesticide technical field, be specifically related to simple genus bacillus and the application of the anti-soybean cyst nematode Heterodera glycines of a kind of tool inducing soybean.
Background technology
Soybean cyst nematode (Soybean Cyst Nematode, be called for short SCN) be by soybean cyst nematode Heterodera glycines (
heterodera glycines) infect and cause, be that harm world soybean is produced one of the most serious disease.This disease has the features such as distribution is wide, harm is heavy, host range is wide, route of transmission is many, hypopus (cyst) survival time is long, is the soil-borne disease of a kind of extremely difficulty control.In recent years along with the continuous expansion of China's soybean culture area, the distribution of soybean cyst nematode Heterodera glycines is also more and more wider, mainly occurs in northeast and Huang-Huai-Hai Deng Gong14Ge provinces, cities and autonomous regions, the general underproduction 20%～30% after soybean is caused harm by it, severe one can reach 70%～80%, and even No kernels or seeds are gathered, as in a year of scarcity.Soybean cyst nematode Heterodera glycines is the settling down property endoparasitism nematode that soil passes, and infects plant root, becomes the main bugbear in the Soybean production of current countries in the world, and most of nematode killing agent serious environment pollutions that chemical prevention is used destroy the eubiosis.Over nearly 20 years, along with the development of sustainable agriculture and Organic farming, people have turned to biological control by the focus of preventing and treating nematodiasiss.
The biological and ecological methods to prevent plant disease, pests, and erosion factor of soybean cyst nematode Heterodera glycines has a lot, comprises fungi, bacterium, virus and protozoon etc.Utilize the existing research of plant rhizosphere growth-promoting bacterium (plant growth promoting rhizobacteria, PGPR) control soybean cyst nematode evil, Becker etc. within 1988, reported Rhodopseudomonas (
pseudomonas) and bacillus (
bacillus) root knot nematode disease is had to preventive and therapeutic effect.Racke and Sikora have reported rhizosphere bacteria for 1992
bacillus sp. and
b.cereusto control Meloidogyne incognita (
meloidogyne incognita), soybean cyst nematode Heterodera glycines (
heterodera glycines), corn Cyst nematode (
heterodera zeae) and Heterodera avenae (
heterodera avenae) larva is effective.Bacillus (
bacillus sp.) bacterium can produce multiple antimicrobial substance, comprise lipopeptid class, peptide class, phospholipid, bacteriophage-like particle and bacteriocin etc., there is the function of antibacterium, fungi, virus and mycoplasma.And genus bacillus is easy to breeding, accommodative ability of environment is strong; Most genus bacillus are nontoxic to people and animals, have very strong environmental compatibility and environment friendly, thereby play an important role in biological control, have very large value of exploiting and utilizing.Correlative study shows that simple genus bacillus has growth-promoting and Biocontrol Effect, and its control rate for soft rot of Chinese cabbage, up to 83%, increases Chinese Cabbage Yield 50% simultaneously, and simple genus bacillus also not yet has report to the control of soybean cyst nematode.
For producing upper this problem that exists, by screening system, obtain a strain bacterium, utilize the meta-bolites processed soybeans seed of this bacterium or this bacterium, can during Seed Germination and growth, produce the resistance to soybean cyst nematode Heterodera glycines by inducing soybean, thereby solve the problem of soybean cyst nematode.
Summary of the invention
After the object of the invention is to select a strain bacterial strain to process soybean seeds, can induce Soybean Seedling to produce obvious resistance to Cyst nematode, by bacterial strain routinely after liquid fermentation and culture, can be with the meta-bolites of this bacterium, develop the seed treatment agent of processed soybeans seed, treated soybean seeds can produce the resistance to soybean cyst nematode Heterodera glycines by inducing soybean.
The strain bacterial isolates that the present invention screens acquisition be simple genus bacillus (
bacillus simplex) Sneb545, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center; Address: No. 1, BeiChen West Road, Chaoyang District, BeiJing City Institute of Microorganism, Academia Sinica; Preservation date: on October 11st, 2012; The numbering that preservation is registered on the books: CGMCC No.6650.Simple genus bacillus (
bacillus simplex) Sneb545 is that the strain filtering out from Chinese Shenyang City, Liaoning Province Agricultural University Of Shenyang experimental plot has the bacterial isolates of strong induced activity to soybean.
The present invention finds by research, and soil bacteria has a lot of strain bacteriums all to have the ability of certain anti-Cyst nematode of inducing soybean, filters out strain proterties bacterial strain relatively preferably from these bacteriums---simple genus bacillus (
bacillus simplex) Sneb545 carried out further research.
The invention still further relates to the bacterium metabolite that produces anti-Cyst nematode resistance for inducing soybean, its be by the simple genus bacillus of bacterial isolates of the present invention (
bacillus simplex) Sneb545 obtains after conventional liquid fermentation and culture.
The simple genus bacillus of bacterial isolates of the present invention (
bacillus simplex) Sneb545 is after cultivating by culture dish, then prepare through expanding fermentation culture, its concrete grammar is as follows:
1. culture dish is cultivated
Culture medium prescription is beef extract-peptone nutrient agar (NA): beef extract 3g, peptone 5g, sucrose 10g, agar 17-20g, distilled water 1000mL.
2. liquid fermentation and culture
Wherein liquid fermentation medium formula is: by weight percentage, yeast extract 0.1%-0.2%, peptone 0.5%-0.6%, beef extract 0.3%-0.4%, sucrose 1%-2%, remainder is distilled water, pH is 6.8-7.0.
Bacterial classification is inoculated in 250mL triangular flask (every bottled 100mL) liquid nutrient medium, and at 25 ℃～28 ℃, shaking speed is with 150r min
-1～180rmin
-1, fermentation 2-3d, preferably 3d.
The bacterial classification of liquid fermenting can carry out scale production by large fermentation tank, and its product is the finished product of fermented liquid or fermented liquid.
The invention still further relates to a kind of soybean seeds treatment agent, its of the present invention simple genus bacillus that comprises significant quantity (
bacillus simplex) Sneb545 itself or its metabolite be as effective active composition.
Simple genus bacillus of the present invention (
bacillus simplex) Sneb545 or its metabolite have the induced activity to soybean, can be used for the seed treatment of soybean, the soybean after processing forms and has good restraining effect the cyst of soybean cyst nematode Heterodera glycines, has solved a difficult problem for soybean cyst nematode.
embodiment:
In order further to illustrate in more detail rather than in order to limit the present invention, to have provided the following example.
embodiment 1: simple genus bacillus (
bacillus simplex) cultivation of Sneb545
First to simple genus bacillus (
bacillus simplex) Sneb545 bacterial strain carries out fermentation culture, then the meta-bolites after fermentation culture carried out to the test of soybean induced activity, determines its inducing action to soybean.
By simple genus bacillus (
bacillus simplex) thalline of Sneb545 is inoculated on substratum, culture medium prescription is beef-protein medium, i.e. composition: beef extract 3g, and peptone 5g, sucrose 10g, agar 17-20g, adding distil water, to 1000mL, is cultivated 2d for 25 ℃.
Again bacterial classification is inoculated in 250mL triangular flask (every bottled 100mL) liquid nutrient medium, culture medium prescription is: by weight percentage, yeast extract 0.1%-0.2%, peptone 0.5%-0.6%, beef extract 0.3%-0.4%, sucrose 1%-2%, remainder is distilled water, pH is 6.8-7.0, leavening temperature is 25-28 ℃, shaking speed 150rmin
-1～180rmin
-1, fermentation time 2-3d.The fermented liquid forming or the finished product of fermented liquid are processed soybeans seed directly or indirectly.Processed soybean seeds can alleviate the harm causing due to soybean cyst nematode evil.
embodiment 2: simple genus bacillus (
bacillus simplex) fermentation of Sneb545
Substantially with embodiment 1, difference is that the fermentor tank that the bacterial classification of liquid culture is inoculated into 1000L ferments, and at 25 ℃, cultivates, and pH is 7.0, fermentation 3d.
embodiment 3: simple genus bacillus (
bacillus simplex) checking of Sneb545 inducing soybean system resistance
Utilization is split root test design (split-root system) and has been inquired into the inducible system resistance of bacterial strain Sneb545 to soybean cyst nematode Heterodera glycines.Split the challenge root system in root test and reply root system spatially isolation completely each other, reply Sneb545 fermented liquid in root system with soybean cyst nematode Heterodera glycines directly contacting, bacterial strain is to the control action kou of nematode its induction of resistance to plant that places one's entire reliance upon.This measuring method is reliable, has been widely used in the system induction resistance research of Biocontrol microorganism to soil-borne disease.
1. prepare test preparation
The fermented liquid of preparing simple Bacillus strain Sneb545 by aforementioned liquids fermentation culture method, treats standby.
2. nematode is used in preparation test
Soybean cyst nematode Heterodera glycines (
heterodera glycines): No. 3 physiological strains of soybean cyst nematode Heterodera glycines are taken from north nematode institute of Agricultural University Of Shenyang test base.Adopt improvement to eluriate-cross sieve method separated cyst from the soil sample of taking, the cyst of fresh, full, ripe, the homogeneous of picking under stereoscope.Cyst is first with the 0.5% chlorine bleach liquor 3min that sterilizes, then uses aseptic water washing 5 times, is placed in the culture dish that contains a small amount of sterilized water, in 25 ℃ of thermostat containers, cultivates, and tray hatching obtained 2 instar larvaes after 15 days, and 2 instar larvae suspension are adjusted to 200/ml.
3. prepare test seed
The Liao Dynasty's beans 15: provided by Liaoning Province Academy of Agricultural Sciences.
4. method for treating seeds
Soybean seeds is first used to 5% chlorine bleach liquor's surface sterilization 5min, then use aseptic water washing 5 times.
5. test method
Employing greenhouse pot culture is processed, and gets without the soil of soybean cyst nematode Heterodera glycines and husky, and in dry sterilization pot 165 ℃, 2h sterilizing, V (soil): the V(of then take is husky) as mixing, the ratio of 2:1 packs in 16 * 16cm polypots.In every alms bowl, sow 3 distant beans 15 seeds, when bean seedlings grow to two cotyledons, every alms bowl stays 1 strain.Bean seedlings are taken out and cleaned, and root system is divided into impartial two portions.Get 3 onesize polypotss, become " product " font to place, the polypots of bottom is put in respectively in the pan arrest of 2 16cm diameters.2 1cm * 2cm are opened in polypots bottom at top, seedling is placed in the polypots at top, make root system separately respectively in the soil of two polypotss by arrival bottom, 2 cracks, the root system of growing in the polypots of two of bottoms is respectively challenge root system and replys root system, packs fixedly plant of soil matrix in 3 alms bowls into.System is placed in greenhouse (25 ℃, 14h illumination), in the polypots of challenge root system, below 2cm table soil, inoculates Sneb545 fermented liquid 10ml, and inoculation Sneb545 fermented liquid is inoculated 10ml J2 suspension to replying root system after 1 week.Take sterilized water as contrast, and totally 2 processing, repeat for 3 times.35d " Invest, Then Investigate " is replied cyst amount and the sum of the soybean cyst nematode Heterodera glycines in root in the soil in root system alms bowl.
The separation of cyst and counting in 5.1 soil: each soil sample takes 200cc (with the 1000mL beaker 800mL that is filled with water, adding soil to 1000mL), adopts the graceful funnel method of eluriate-sieve-shellfish to collect cyst, records the quantity of cyst under stereoscopic anatomical lens.
The dyeing of 5.2 interior nematodes and counting: root system is cleaned up with flowing water, claim root fresh weight, then use clorox-acid fuchsin staining to carry out the dyeing of nematode in root, nematode population in observed and recorded root under stereoscopic microscope, and be converted into the quantity of every gram of root fresh weight nematode.
5.3 prevention effect are calculated
Cyst inhibiting rate (%)=
Nematode decrement (%)=
6 test-results
Measure and show, Sneb545 can produce the resistance very strong to soybean cyst nematode Heterodera glycines by inducing soybean.Bacterial strain Sneb545 after inoculation, replys the invasion of soybean cyst nematode Heterodera glycines in root system and significantly reduces in challenge root system, replys cyst quantity in Soil-in-Root and significantly reduces.Reply the invasion of root system soybean cyst nematode Heterodera glycines and reduce by 51.27%, in soil, cyst reduces 65.82%(table 1).
After table 1 Sneb545 inoculation challenge root system, reply and in root system, invade cyst sum in nematode and soil
| Process | Cyst quantity in soil | Soybean cyst nematode Heterodera glycines quantity in root |
| Sneb545 | 9.0000±2.6458bB | 32.0000±4.3589bB |
| CK | 26.3333±2.6034aA | 65.6667±5.6075aA |
Note: the investigation result after 35 d, after numeral, letter is Duncan ' s multiple range test result, different capital and small letters are English
Letter represents respectively difference extremely significantly (P < 0. 01) and significant difference (P < 0. 05).
embodiment 4: simple genus bacillus (
bacillus simplex) the field control soybean cyst nematode effect test of Sneb545
Use fermented liquid stoste, in the ratio treatment coatings soybean seeds of 1:100, after drying, the soybean seeds of processing is normally sowed on soybean cyst nematode piece ground, after Sneb545 metabolite processed soybeans seed, can induce Soybean Seedling to produce obvious resistance to Cyst nematode, to the obvious restraining effect of being formed with of soybean cyst nematode Heterodera glycines cyst, be very potential biocontrol microorganisms, for control soybean cyst nematode has been opened up new approaches.
1. prepare test preparation
The fermented liquid of preparing simple Bacillus strain Sneb545 by aforementioned liquids fermentation culture method, treats standby.
2. prepare test seed
The Liao Dynasty's beans 15: provided by Liaoning Province Academy of Agricultural Sciences.
Close rich 50: by grand celebration branch of Heilongjiang Institute of Agricultural Sciences, provided.
3. method for treating seeds
Soybean seeds is first used to 5% chlorine bleach liquor's surface sterilization 5min, then use aseptic water washing 5 times.The grain weight of the fermented liquid employing 1% preparing is carried out to seed pelleting processing, standby after drying.
4. test method
Adopt field natural occurrence soil inoculation soybean.In Kangping County, Liaoning Province and two places, Daqing, Heilongjiang Province, carry out revision test 5 times respectively.Soybean Field is secondary soybean field, test base, Kangping continuous cropping 3 years, and field cyst amount is 40 cyst/100ml soil, and grand celebration test base continuous cropping is more than 6 years, and field cyst amount is 55 cyst/100ml soil.Field Quarter Design is 3 processing, repeats for 5 times, and Gong15Ge community, random district group is arranged 5 ridges, ，Mei community, long 5 meters of ridge, 1 meter, passageway, 100 of every ridge plantings, cell edge is established protection row.Take is not contrast through the soybean seeds of dressing and the soybean seeds of NA liquid medium dressing, 35d after soybean planting, until soybean cyst nematode Heterodera glycines, grow and break through after soybean rhizoplane, each community is got 20 young plants at random, after removing table soil (0-5cm), plant is grubbed out out, keep the complete of root system, investigation individual plant soybean root system cyst raw amount, calculates cyst inhibiting rate.
5. test-results
The soybean that Sneb545 bacterial strain is processed, at the aobvious capsule phase investigation of soybean cyst nematode Heterodera glycines root system cyst, raw situation, large bean seedlings after Sneb545 bacterial isolates Cotton seeds have formed obvious restraining effect to the cyst of soybean cyst nematode Heterodera glycines, as shown in Table 2, Sneb545 has reached respectively 57.67%, 47.92% at the inhibiting rate of large Khanh Hoa Kangping, soybean after Cotton seeds has different in the result of Kangping and the processing of grand celebration two places, and this may be relevant with the difference of different areas Soybean Cyst Nematode.
The impact that table 2 Sneb545 bacterial strain Cotton seeds soybean seeds forms soybean root system cyst
| Process | Heilungkiang grand celebration | Kangping, Liaoning |
| Sneb545 | 39.2±3.6387bBC | 45.35±4.7315bcdB |
| NA | 93.2±5.0735aA | 84.34±5.8726aA |
| CK | 92.6±3.7762aA | 87.08±8.1215aA |
Note: the investigation result after 35 d, after numeral, letter is Duncan ' s multiple range test result, different capital and small letters are English
Letter represents respectively difference extremely significantly (P < 0. 01) and significant difference (P < 0. 05).
Claims (7)
1. after a strain processed soybeans seed, there is the bacterial isolates of the anti-soybean cyst nematode Heterodera glycines of inducing soybean, it is characterized in that: this bacterial strain be simple genus bacillus (
bacillus simplex), on October 11st, 2012, being deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.6650.
2. the liquid fermentation process of bacterial isolates claimed in claim 1, it is characterized in that: liquid fermentation medium formula is: by weight percentage, yeast extract 0.1%-0.2%, peptone 0.5%-0.6%, beef extract 0.3%-0.4%, sucrose 1%-2%, remainder is distilled water, pH is 6.8-7.0, leavening temperature is 25-28 ℃, shaking speed 150rmin
-1～180rmin
-1, fermentation time 2-3d.
3. liquid fermentation process according to claim 2, is characterized in that: described fermentation time is 3d.
4. there is the preparation method that inducing soybean produces the fermented liquid stoste of anti-soybean cyst nematode Heterodera glycines resistance, it is characterized in that: by bacterial isolates claimed in claim 1, after liquid fermenting, formed, liquid fermentation medium formula is: by weight percentage, yeast extract 0.1%-0.2%, peptone 0.5%-0.6%, beef extract 0.3%-0.4%, sucrose 1%-2%, remainder is distilled water, pH is 6.8-7.0, and leavening temperature is 25-28 ℃, shaking speed 150rmin
-1～180rmin
-1, fermentation time 2-3d.
5. preparation method according to claim 4, is characterized in that: described fermentation time is 3d.
6. a soybean seeds treatment agent, is characterized in that, formed fermented liquid stoste in the liquid fermentation process according to claim 2 that comprises significant quantity produces the resistance to soybean cyst nematode Heterodera glycines for inducing soybean.
7. the application of the fermented liquid stoste forming in liquid fermentation process described in claim 2 in the anti-soybean cyst nematode Heterodera glycines of inducing soybean.
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| CN105002121B (en) * | 2015-08-14 | 2018-02-23 | 山东泰诺药业有限公司 | One plant of simple bacillus and its application |
| CN105112340B (en) * | 2015-09-15 | 2018-08-28 | 沈阳农业大学 | One kind having the active bacterium bacterial strain of induction of resistance and its cultural method and application |
| CN105494447B (en) * | 2015-12-23 | 2018-11-02 | 沈阳农业大学 | Biological seed coating agent and its preparation method and application for controlling soybean root disease |
| CN109769858B (en) * | 2016-04-25 | 2020-12-11 | 北京市农林科学院 | Plant root-knot nematode inhibitor and application thereof |
| CN106986719A (en) * | 2016-10-26 | 2017-07-28 | 沈阳农业大学 | And anti-root rot and seed coat agent, the preparation method of soybean cyst nematode |
| CN110275001B (en) * | 2019-07-31 | 2023-01-03 | 四川省农业科学院生物技术核技术研究所 | Device and method for measuring and evaluating drug effect of drug on root-knot nematodes |
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| CN102308853A (en) * | 2011-03-02 | 2012-01-11 | 沈阳农业大学 | The biological seed coating agent of control soybean SCN |
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