CN104946551A - Bacillus strain for controlling soybean root rot and Bacillus microbial inoculant thereof - Google Patents

Bacillus strain for controlling soybean root rot and Bacillus microbial inoculant thereof Download PDF

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CN104946551A
CN104946551A CN201510019431.7A CN201510019431A CN104946551A CN 104946551 A CN104946551 A CN 104946551A CN 201510019431 A CN201510019431 A CN 201510019431A CN 104946551 A CN104946551 A CN 104946551A
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bacillus
gemma
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root rot
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CN104946551B (en
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魏利辉
陈曦
黄慧文
卢晓雪
冯辉
张金凤
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Jiangsu Academy of Agricultural Sciences
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Abstract

The invention discloses a Bacillus strain for controlling soybean root rot, which is Bacillus.sp. The collection name is Bacillus JSCX-1; the Bacillus strain is collected by China General Microbiological Culture Collection Center (CGMCC); the collection address is Yard 1 No.3, Beichenxi Road, Chaoyang District, Beijing City; and the collection date is January 7th, 2015; and the collection number is CGMCC No.10284. The single bacterial colony of the Bacillus strain is circular and milk white, the long-term cultured bacterial colony is slightly brown yellow and nontransparent, has regular edges, and has slight wrinkles on the surface. The Bacillus strain is purple by Gram staining, and is positive. In spore staining, the spore is green, and the thallus is red. The Bacillus strain grows along the culture medium surface in an aerobic test, and thus, is an aerobic bacterium. The Bacillus strain for controlling soybean root rot and Bacillus microbial inoculant thereof can not easily enable soybean phytophthora to generate resistance, and can not easily pollute the environment.

Description

Prevent and treat Bacillus strain and the gemma bacillus agent thereof of root rot
Technical field
The present invention relates to a kind of microorganism field, be specifically related to a kind of Bacillus strain and the gemma bacillus agent thereof of preventing and treating root rot.
Background technology
It is a kind of destructive disease for Soybean production that soybean phytophthora infects the root rot that soybean plant strain causes, this disease found in the U.S. early than 1948, constantly spread to nearly 20 the national soybean producing regions in Asia, non-, Europe, the United States continent afterwards, the direct economic loss brought every year, up to tens dollars, is one of disease the most serious in Soybean production.At the end of the eighties, this disease finds in the Northeast of China first, has become the No.1 disease that area of Northeast is produced, has caused huge financial loss.
Phytophthora sojae kaufmann&gerdemann can work the mischief to soybean in each breeding time of soybean.Seed decay can be caused before soybean emerges, emerge before rot, can cause after emerging plant wither.Disease plant showed as poor, the near surface stem of emerging and occurred that water soaking mode scab, blade turn yellow and wilt general seedling stage, seriously entered to plant death of dampinging off.Adult plant plant is infected rear lower blade and is turned yellow, and upper blade turns yellow gradually and wilts very soon subsequently; Near surface stem scab brown, and can upwards expand, stem cortex and marrow browning; Root rot, root system development is bad; The number that bears pods of not dead diseased plant obviously reduces, and empty pod, flat pod are more, seed shrinkage.Due in humid conditions, the germ that root infects can produce a large amount of zoospore, spore splashes with rainwater, harm stem and blade, even occur disease pod, its symptom is that water soaking mode spot appears in green beanpod base portion, scab gradually browning and from pod handle spread to pod point, last whole beanpod is that tawny dries up, and seed dehydration is shrivelled.
Current soybean blight mainly passes through chemical prevention.Although chemopreventive effects is remarkable, because its action site is single, the resistance problems that life-time service makes soybean phytophthora produce becomes increasingly conspicuous.The biological activity of such as the most frequently used medicament metaxanin (trade name Ridomil) is strong, has lasting sterilization effect.But the action site of metaxanin to soybean phytophthora is single, the sudden change of the soybean phytophthora occurred at this action site all likely can produce resistance to medicament.In addition the poor selectivity of chemical prevention, the ecotope of destroy crops, not easily degrades, and is absolutely unsafe to people and animals.The restriction that these factors make the reagent use range of chemical prevention be subject to.Therefore, developing the measure that non-chemically prevents and treats has become the emphasis that soybean phytophthora administers at present, and to carry out biological control with gemma bacillus agent be wherein the most promising method.
At present, lack a kind of soybean phytophthora that not easily makes and produce resistance, the free from environmental pollution Bacillus strain preventing and treating root rot and gemma bacillus agent thereof.
Summary of the invention
Technical problem to be solved by this invention there is provided and a kind ofly not easily makes epidemic disease mould generation resistance, the free from environmental pollution Bacillus strain preventing and treating root rot and gemma bacillus agent thereof.
To achieve these goals, the present invention is achieved through the following technical solutions: a kind of Bacillus strain preventing and treating root rot, and this bacterial strain is genus bacillus (BaciLLus.sp); Preservation name is called genus bacillus JSCX-1; Be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Preservation date: on 01 07th, 2015; Deposit number: CGMCC No.10284.
Further, single bacterium colony of described Bacillus strain is circular, oyster white, the inclined brown color of long-term cultivation bacterium colony, neat in edge, and opaque, surface is fold slightly.
Further, described Bacillus strain gramstaining is purple, is the positive; In spore staining, gemma is in green, and thalline takes on a red color; Bacillus strain is along media surface growth in aerobic-type test-results, is aerobic bacteria.
The gemma bacillus agent of root rot that prepared by genus bacillus of the present invention prevent and treat.
Further, the described gemma bacillus agent preventing and treating root rot, its activeconstituents is at least one in following (a), (b), (c) and (d):
The fermenting culture of genus bacillus described in (a) claim 1;
Genus bacillus described in (b) claim 1;
The gemma of genus bacillus described in (c) claim 1;
The fermentation supernatant of genus bacillus described in (d) claim 1.
The preparation method preventing and treating the gemma bacillus agent of root rot of the present invention, comprises the steps:
(1) be bacillus by described Bacillus strain JSCX-1, Bacillus in NB substratum on growth, temperature is 29-31 DEG C, vibrate under rotating speed is 150-180r/min and cultivate, incubation time is 16-20h, then in Bechtop, OD600 value is surveyed in sampling respectively, compares in mensuration process with the NB substratum not connecing bacterium;
(2) when Bacillus is cultured to OD600 value for 0.5-0.8, Bacillus solution and distilled water are added in NB substratum according to the volume ratio of 1:100, temperature are 28-30 DEG C, rotating speed is shaking culture 20-24h under 150-180r/min,
Then under rotating speed is 6000-8000r/min, centrifugation 9-11min is carried out, obtained gemma bacillus agent.
Further, in step (2), by the concentration dilution to 1 of gemma bacillus agent × 10 8-1 × 10 10cFU/ml.
Further, in step (1), described NB substratum is by beef extract 2.9-3.1g, and peptone 9.9-10.1g, sodium-chlor 4.9-5.1g form; PH is 6.8-7.0, with distilled water, said components is settled to 1000ml, sterilizing 19-21min at temperature is 120-121 DEG C.
Gemma bacillus agent of the present invention is preventing and treating the application of root rot.
Beneficial effect: the present invention not easily makes soybean phytophthora produce resistance, the free from environmental pollution Bacillus strain preventing and treating root rot and gemma bacillus agent thereof.The problems such as gemma bacillus agent belongs to biotechnological formulation, the chemical resistance not having the use because of chemical agent to bring completely, are thus conducive to the No-harmful apple orchard of soybean.Peasant or can reduce the consumption of the agricultural chemicals of other control soybean phytophthoras.Reduce peasant to reduce expenses.Meanwhile, the prevention effect of this gemma bacillus agent can reach more than 50%, increases production function in addition, can be peasant and increases income.Gemma bacillus agent is 55-64% to the prevention effect of root rot, and agricultural produce can reach more than 23%.
Accompanying drawing explanation
Fig. 1: JSCX-1 genome and the higher phyletic evolution tree graph constructed by strain sequence of homology.
Embodiment
Further specific descriptions will be done by specific embodiment to the present invention below, but can not be interpreted as it is limiting the scope of the present invention.
Embodiment 1
The physiological and biochemical test of bacterial strain and the qualification of 16S rDNA molecule
As shown in Figure 1, Bacillus strain JSCX-1 carries out activation culture on NA flat board at temperature is 29-31 DEG C, NA substratum: beef extract (Beef Extract) 3g, peptone (Peptone) 10g, sodium-chlor (NaCl) 5g, agar powder (Agar) 15g, pH7.0.
Incubation time is observe after 20-24h to find, single bacterium colony subcircular of JSCX-1, oyster white, the inclined brown color of long-term cultivation bacterium colony, and neat in edge, opaque, surface is fold slightly.Bacterial strain JSCX-1 gramstaining is purple, is the positive; In spore staining, gemma is in green, and thalline takes on a red color.Bacterial strain JSCX-1 is along media surface growth in aerobic-type test-results, shows that bacterial strain JSCX-1 is aerobic bacteria.
Physiological and biochemical test is oxidase test, hydrogen peroxide test, gelatin liquification test, citrate test, H 2the result that S produces test is the positive; Phenylalanine ammonia lyase test, Starch Hydrolysis test, indole test, methyl red test, voges-Proskauer test, nitrate reduction test, in salt tolerance test, result is feminine gender.
Bacillus strain JSCX-1 can containing 2%, 5%, grow in the NA substratum of 8%NaCl, cannot grow in containing the NA substratum of 10%NaCl, sugar-fermenting liquid nutrient medium flavescence look and inverted moral Han Shi tubule has gas to produce in glucose, mannose ferment test.The morphological specificity of bacterial strain JSCX-1 is the most similar to bacillus (Bacillus sp.) in common bacteria system identification handbook with physiological and biochemical property.
With JSCX-1 genome DNA for template, pcr amplification 16SrDNA obtains the amplified production that length is about 1.5Kb, and this and 16SrDNA most high conservative and consistent length in all bacteriums are that the report of about 1.5Kb coincide.The total length that arrangement sequencing result can obtain the 16SrDNA sequence of carrying out Phylogenetic Analysis is 1419bp, by sequencing result Blast search in the GenBank database of NCBI website, carry out nucleotide homology with listed 16SrDNA sequence to compare, it is higher that the 16SrDNA sequence of discovery bacterial strain JSCX-1 and many bacillus belong to bacterium (Bacillus sp.) similarity, download the sequence higher with the 16SrDNA sequence homology of Bacillus strain JSCX-1, choose the constructing system evolutionary tree that 10 strain similarities are the highest, find that bacterial strain JSCX-1 and Bacillus sp.Q2-S1 (JX994136.1) is in same branch, homology is the highest, be greater than 99%, in conjunction with cultural characters and physiological and biochemical property qualification result, Bacillus strain JSCX-1 is initially identified as bacillus (Bacillus sp.)
The present invention not easily makes soybean phytophthora produce resistance, the free from environmental pollution Bacillus strain preventing and treating root rot and gemma bacillus agent thereof.The problems such as gemma bacillus agent belongs to biotechnological formulation, the chemical resistance not having the use because of chemical agent to bring completely, are thus conducive to the No-harmful apple orchard of soybean.Peasant or can reduce the consumption of the agricultural chemicals of other control soybean phytophthoras.Reduce peasant to reduce expenses.Meanwhile, the prevention effect of this gemma bacillus agent can reach more than 50%, increases production function in addition, can be peasant and increases income.Gemma bacillus agent is 55-64% to the prevention effect of root rot, and agricultural produce can reach more than 23%.
Embodiment 2
Soybean phytophthora zoospore suspension preparation method step is as follows:
(1) the soybean phytophthora bacterial strain activated growth on V8 substratum will preserved, the activated growth time is 5-6d.
(2) tomato and lettuce are cleaned dry with organizing pulper to blend.Remove slag by four layers of filtered through gauze.Mix according to the ratio of tomato juice and lettuce juice 1:2.
(3) CaCO is added in the ratio of 0.02% (w/v) 3, then add the vegetable juice culture that deionized water is mixed with 50%.Temperature is 120-122 DEG C, and sterilization time is 29-31min.
(4) by activation soybean phytophthora bacterial strain on LBA substratum, temperature is 24-26 DEG C, and dark condition is cultivated, and incubation time is get bacterium dish with punch tool (d=5mm) along colony edge after 3-4d.
(5) 4 ferfas dish are placed in 14-16ml substratum, 24-26 DEG C, dark condition is cultivated, and incubation time is 3-4d.
(6) incline substratum, adds the aqua sterilisa that volume is 19-21ml, and interval time is that 12-14h changes an aqua sterilisa again.
(7) at temperature is 24-26 DEG C, constant temperature illumination box is cultivated, and incubation time is 20-24h, produces zoosporangium.
(8) zoosporangium suspension is carried out homogenate 1min under rotating speed is 3000-3200r/min, be adjusted to concentration with sterilized water and be about 1 × 10 4-1 × 10 5cFU/ml.
A kind of Bacillus strain preventing and treating root rot of the present invention, this bacterial strain is genus bacillus (BaciLLus.sp); Preservation name is called genus bacillus JSCX-1; Be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Preservation date: on 01 07th, 2015; Deposit number: CGMCC No.10284.
Single bacterium colony of described Bacillus strain is circular, oyster white, the inclined brown color of long-term cultivation bacterium colony, neat in edge, and opaque, surface is fold slightly.Described Bacillus strain gramstaining is purple, is the positive; In spore staining, gemma is in green, and thalline takes on a red color; Bacillus strain is along media surface growth in aerobic-type test-results, is aerobic bacteria.
The gemma bacillus agent of root rot that prepared by genus bacillus of the present invention prevent and treat.Its activeconstituents is at least one in following (a), (b), (c) and (d):
The fermenting culture of genus bacillus described in (a) claim 1;
Genus bacillus described in (b) claim 1;
The gemma of genus bacillus described in (c) claim 1;
The fermentation supernatant of genus bacillus described in (d) claim 1.
The preparation method preventing and treating the gemma bacillus agent of root rot of the present invention, comprises the steps:
(1) be bacillus by described Bacillus strain JSCX-1, Bacillus in NB substratum on growth, temperature is 29-31 DEG C, vibrate under rotating speed is 150-180r/min and cultivate, incubation time is 16-20h, then in Bechtop, OD600 value is surveyed in sampling respectively, compares in mensuration process with the NB substratum not connecing bacterium; Described NB substratum is by beef extract 2.9-3.1g, and peptone 9.9-10.1g, sodium-chlor 4.9-5.1g form; PH is 6.8-7.0, with distilled water, said components is settled to 1000ml, sterilizing 19-21min at temperature is 120-121 DEG C.
(2) when Bacillus is cultured to OD600 value for 0.5-0.8, Bacillus solution and distilled water are added in NB substratum according to the volume ratio of 1:100, temperature is 28-30 DEG C, rotating speed is shaking culture under 150-180r/min, incubation time is 20-24h, then under rotating speed is 6000-8000r/min, centrifugation is carried out, disengaging time is 9-11min, obtained gemma bacillus agent.By the concentration dilution to 1 of gemma bacillus agent × 10 8-1 × 10 10cFU/ml.
Gemma bacillus agent of the present invention is preventing and treating the application of root rot.
Excised leaf prevention effect measures
Gemma bacillus agent is diluted to predetermined concentration for subsequent use; This microbial inoculum can fill with root use or seed soaking use before sowing by 2-3d after emerging.From growth neat soybean plant strain gather healthy leaves of the same size, rinse well under tap water, with 70% alcohol-pickled, soak time is 30s, dries in Bechtop with after aseptic water washing 2min.Soybean leaves is immersed in wherein 1min, takes out and dry in Bechtop.Blade connects 1 μ L soybean phytophthora zoospore suspension, blade to connect NB substratum for contrast, each process repetition 5 times.At 24-26 DEG C, relative humidity 80-90%, dark condition measures lesion diameter after cultivating 3-4d, assessment preventive effect.The calculation formula of preventive effect is: preventive effect (%)=(control group lesion diameter-treatment group lesion diameter)/control group lesion diameter × 100%.
Table 1 is that the gemma bacillus agent of genus bacillus JSCX-1 measures the prevention effect of soybean excised leaf.
Table 1
As table 1 can draw, soybean excised leaf experimental result shows that concentration is 10 8-10the JSCX-1 gemma bacillus agent of CFU/ml has good restraining effect to soybean leaves being inoculated soybean phytophthora zoospore.After 3-4d, the preventive effect of JSCX-1 gemma bacillus agent to soybean phytophthora zoospore reaches 64.21%.Excised leaf experimental result shows that the gemma bacillus agent of genus bacillus JSCX-1 is 1 × 10 8-1 × 10 10the concentration of CFU/ml has good restraining effect to soybean phytophthora.Letter representations different in table is significant difference in 0.05 level.
Embodiment 3
Potted plant prevention effect measures
Embodiment 3 is with the difference of embodiment 2: fill with as genus bacillus bacterium gemma bacillus agent to the soybean seedling root growing 2 true leaves, and 15 ± 1ml is filled with in every strain.The seedling of isopyknic NB substratum is poured into for contrast with root position.Fill with soybean phytophthora zoospore suspension to soybean seedling root after 3-4d, every strain pours into 30 ± 1ml.Each process repetition 5 times.24-26 DEG C of cultivation, after 9-11d, calculate sickness rate, disease index and prevention effect.Each parameter calculation formula is as follows:
Sickness rate (%)=(disease plant number/total plant number) × 100%
Disease index=100 × ∑ (severity plant number × typical value at different levels at different levels)/(investigation plant number × highest typical value)
Prevention effect (%)=[(control group disease index-treatment group disease index)/control group disease index] × 100%
Table 2 is that the potted plant prevention effect of genus bacillus JSCX-1 gemma bacillus agent to soybean blight measures;
Table 2
As table 2 can draw, detect after 9-11 days at inoculation soybean phytophthora zoospore liquid, potted plant experiment result shows the gemma bacillus agent leaching root process through genus bacillus JSCX-1, and soybean plant strain effectively can resist the infection of soybean blight, and preventive effect reaches 54.59%; Letter representations different in table is significant difference in 0.05 level.
Embodiment 4
To the growth-promoting effect measuring of soybean seedling
Embodiment 4 is with the difference of embodiment 2: pour into 15 ± 1ml genus bacillus bacterium gemma bacillus agent to the soybean seedling root growing 4 leaves, pour into the seedling of isopyknic NB substratum in contrast with root, each process repetition 3 times.Measure the fresh weight of large bean seedlings, dry weight after 15 ± 1d, root is long and stem is long, calculates biomass increment rate.The calculation formula of biomass increment rate is: biomass increment rate (%)=(treatment group mean fresh-control group mean fresh)/control group mean fresh × 100%.
Table 3 is the growth-promoting effect measuring to soybean seedling;
Table 3
As table 3 can draw, after growth-promoting effect experimental shows the gemma bacillus agent root irrigation 15 ± 1d with genus bacillus JSCX-1, large bean seedlings indices increases all to some extent.Average every young plant fresh weight increases by 37.06%, dry weight increases by 23.53%, the long increase by 26.65% of root, the long increase by 11.31% of stem, Biological control result shows that the every growth indexes of large bean seedlings increases all to some extent after the gemma bacillus agent root irrigation of genus bacillus JSCX-1.Letter representations different in table is significant difference in 0.05 level;
Can prove according to above each embodiment: the medicament being effective constituent with gemma bacillus agent of the present invention is a kind of novel agricultural bacteriocide.This medicament particularly demonstrates remarkable effect to root rot, and environmental pollution is little, the feature that resistance is low.
More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and application claims protection domain is defined by appending claims, specification sheets and equivalent thereof.

Claims (9)

1. prevent and treat a Bacillus strain for root rot, it is characterized in that: this bacterial strain is genus bacillus (BaciLLus.sp); Preservation name is called genus bacillus JSCX-1; Be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Preservation date: on 01 07th, 2015; Deposit number: CGMCC No.10284.
2. the Bacillus strain preventing and treating root rot according to claim 1, is characterized in that: single bacterium colony of described Bacillus strain is for circular, and oyster white, the inclined brown color of long-term cultivation bacterium colony, neat in edge, opaque, surface is fold slightly.
3. the Bacillus strain preventing and treating root rot according to claim 2, is characterized in that: described Bacillus strain gramstaining is purple, is the positive; In spore staining, gemma is in green, and thalline takes on a red color; Bacillus strain is along media surface growth in aerobic-type test-results, is aerobic bacteria.
4. the gemma bacillus agent preventing and treating root rot that prepared by genus bacillus according to claim 1.
5. the gemma bacillus agent preventing and treating root rot according to claim 4, its activeconstituents is at least one in following (a), (b), (c) and (d):
The fermenting culture of genus bacillus described in (a) claim 1;
Genus bacillus described in (b) claim 1;
The gemma of genus bacillus described in (c) claim 1;
The fermentation supernatant of genus bacillus described in (d) claim 1.
6. prepare the method for preventing and treating the gemma bacillus agent of root rot described in claim 1 or 2, it is characterized in that comprising the steps:
(1) be bacillus by described Bacillus strain JSCX-1, Bacillus in NB substratum on growth, temperature is 29-31 DEG C, vibrate under rotating speed is 150-180r/min and cultivate, incubation time is 16-20h, then in Bechtop, OD600 value is surveyed in sampling respectively, compares in mensuration process with the NB substratum not connecing bacterium;
(2) when Bacillus is cultured to OD600 value for 0.5-0.8, Bacillus solution and distilled water are added in NB substratum according to the volume ratio of 1:100, temperature are 28-30 DEG C, rotating speed is shaking culture 20-24h under 150-180r/min,
Then under rotating speed is 6000-8000r/min, centrifugation 9-11min is carried out, obtained gemma bacillus agent.
7. the preparation method of gemma bacillus agent according to claim 6, is characterized in that: in step (2), by the concentration dilution to 1 of gemma bacillus agent × 10 8-1 × 10 10cFU/ml.
8. the preparation method of gemma bacillus agent according to claim 6, is characterized in that: in step (1), and described NB substratum is by beef extract 2.9-3.1g, and peptone 9.9-10.1g, sodium-chlor 4.9-5.1g form;
PH is 6.8-7.0, with distilled water, said components is settled to 1000ml, sterilizing 19-21min at temperature is 120-121 DEG C.
9. gemma bacillus agent described in claim 4 ~ 5 is preventing and treating the application of root rot.
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Cited By (1)

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CN105385623A (en) * 2015-11-20 2016-03-09 江苏省农业科学院 Actinomycete strain JSX-1 for preventing and curing phytophthora sojae and fungicide thereof

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