CN104946551B - The Bacillus strain and its gemma bacillus agent for preventing and treating root rot - Google Patents

The Bacillus strain and its gemma bacillus agent for preventing and treating root rot Download PDF

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CN104946551B
CN104946551B CN201510019431.7A CN201510019431A CN104946551B CN 104946551 B CN104946551 B CN 104946551B CN 201510019431 A CN201510019431 A CN 201510019431A CN 104946551 B CN104946551 B CN 104946551B
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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 kind of Bacillus strain for preventing and treating root rot, which is bacillus (BaciLLus.sp)；The entitled bacillus JSCX-1 of preservation；It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3；Preservation date: on 01 07th, 2015；Deposit number: CGMCC No.10284.The single colonie of the Bacillus strain is circle, and milky, the inclined brown color of long-term cultivation bacterium colony, neat in edge is opaque, and fold is omited on surface.The Bacillus strain Gram's staining is purple, for the positive；Gemma takes on a red color in green, thallus in spore staining；Bacillus strain is to grow along media surface in aerobic-type test result, is aerobic bacteria.The present invention is not easy the Bacillus strain and its gemma bacillus agent for so that soybean phytophthora is generated resistance, prevention and treatment root rot free from environmental pollution.

Description

The Bacillus strain and its gemma bacillus agent for preventing and treating root rot
Technical field
The present invention relates to a kind of microorganism fields, and in particular to it is a kind of prevent and treat root rot Bacillus strain and its Gemma bacillus agent.
Background technique
It is a kind of destructive disease for Soybean production that soybean phytophthora, which infects root rot caused by soybean plant strain, This disease was most found earlier than 1948 in the U.S., constantly spread to sub-, non-, the national soybean producing region in nearly 20 of Europe, the United States continent afterwards, Annual bring direct economic loss is up to tens dollars, is one of most serious disease in Soybean production.It the end of the eighties, should Disease is found in the Northeast of China for the first time, it has also become the No.1 disease in area of Northeast production causes huge warp Ji loss.
Phytophthora sojae kaufmann&gerdemann can cause damages to soybean in each breeding time of soybean.It can cause before soybean emergence It rots before seed decay, emergence, can cause plant withered after emergence.General seedling stage disease plant shows as poor, near-earth of emerging Water soaking mode scab, which occurs, in table stem, blade turns yellow wilts, and seriously dampings off death into plant.Adult plant plant is infected rear lower blade Turn yellow, subsequent upper blade gradually turns yellow and wilts quickly；Near surface stem scab brown, and can extending up, stem cortex and Marrow browning；Root rot, root system development are bad；The number that bears pods of not dead diseased plant significantly reduces, and empty pod, flat pod are more, seed shrinkage. Since in humid conditions, the germ that root is infected can produce a large amount of zoospores, spore splashes with rainwater, endanger stem and Blade, or even sick pod occur, symptom are that green beanpod base portion water soaking mode spot occurs, and scab gradually browning and is spread to from pod handle Pod point, last entire beanpod is withered in yellowish-brown, and seed dehydration is shrivelled.
Soybean blight mainly passes through chemical prevention at present.Although chemopreventive effects are significant, due to its action site Single, the resistance problems for generating soybean phytophthora become increasingly conspicuous.Such as most common medicament metalaxyl (trade name Claim Ridomil) bioactivity it is strong, have lasting bactericidal effect.But metalaxyl is single to the action site of soybean phytophthora, The mutation for the soybean phytophthora that this action site occurs can be likely to generate resistance to medicament.Furthermore the selectivity of chemical prevention Difference destroys the ecological environment of crops, not degradable, is absolutely unsafe to people and animals.These factors make the reagent of chemical prevention With range by being limited.Therefore, the measure for developing non-chemical prevention and treatment has become the emphasis of soybean phytophthora improvement at present, and uses bud Spore bacillus microbial inoculum is come to carry out biological control be wherein most promising method.
Currently, lacking a kind of gemma for being not easy that soybean phytophthora is made to generate resistance, prevention and treatment root rot free from environmental pollution Bacillus strain and its gemma bacillus agent.
Summary of the invention
It is not easy to make phytophthora to generate resistance, free from environmental pollution anti-technical problem to be solved by the invention is to provide a kind of Control the Bacillus strain and its gemma bacillus agent of root rot.
To achieve the goals above, the present invention is achieved through the following technical solutions: a kind of gemma for preventing and treating root rot Bacillus strain, the bacterial strain are bacillus (BaciLLus.sp)；The entitled bacillus JSCX-1 of preservation；It is micro- to be preserved in China Biological inoculum preservation administration committee common micro-organisms center (CGMCC), preservation address are BeiChen West Road, Chaoyang District, BeiJing Cities 1 Institute 3；Preservation date: on 01 07th, 2015；Deposit number: CGMCC No.10284.
Further, the single colonie of the Bacillus strain is round, milky, the inclined brown color of long-term cultivation bacterium colony, Neat in edge, opaque, fold is omited on surface.
Further, the Bacillus strain Gram's staining is purple, for the positive；Gemma is in green in spore staining Color, thallus take on a red color；Bacillus strain is to grow along media surface in aerobic-type test result, is aerobic bacteria.
The gemma bacillus agent of the prevention and treatment root rot of bacillus preparation of the present invention.
Further, the gemma bacillus agent of the prevention and treatment root rot, active constituent is following (a), (b), (c) and at least one of (d):
(a) fermentation culture medium of bacillus described in claim 1；
(b) bacillus described in claim 1；
(c) gemma of bacillus described in claim 1；
(d) fermentation supernatant of bacillus described in claim 1.
The preparation method of the gemma bacillus agent of prevention and treatment root rot of the present invention, includes the following steps:
It (1) is bacillus, Bacillus upper life in NB culture medium by the Bacillus strain JSCX-1 Long, temperature is 29-31 DEG C, vibrates and is cultivated in the case where revolving speed is 150-180r/min, incubation time 16-20h, then super Separately sampled survey OD600 value in net workbench is compared in continuous mode with not connecing the NB culture medium of bacterium；
(2) when Bacillus culture to OD600 value is 0.5-0.8, by Bacillus solution and distilled water It is added in NB culture medium according to the volume ratio of 1:100, temperature is 28-30 DEG C, revolving speed is shaken cultivation under 150-180r/min 20-24h,
Then 9-11min is centrifuged in the case where revolving speed is 6000-8000r/min, gemma bacillus agent is made.
Further, in step (2), by the concentration dilution of gemma bacillus agent to 1 × 108-1×1010CFU/ml。
Further, in step (1), the NB culture medium by beef extract 2.9-3.1g, peptone 9.9-10.1g, Sodium chloride 4.9-5.1g composition；PH is 6.8-7.0, and said components are settled to 1000ml with distilled water, is 120- in temperature Sterilize 19-21min at 121 DEG C.
Application of the gemma bacillus agent of the present invention in prevention and treatment root rot.
The utility model has the advantages that the present invention is not easy the bud for making soybean phytophthora generate resistance, prevention and treatment root rot free from environmental pollution Spore bacillus strain and its gemma bacillus agent.Gemma bacillus agent belongs to biological agent, absolutely not because of the use of chemical agent The problems such as brought chemistry drug resistance, thus be conducive to the No-harmful apple orchard of soybean.It is anti-that peasant can not have to or reduce other Control the dosage of the pesticide of soybean phytophthora.Peasant is reduced to reduce expenses.Meanwhile the control efficiency of the gemma bacillus agent is up to 50% More than, there are also function is increased production, income can be increased for peasant.Gemma bacillus agent is 55- to the control efficiency of root rot 64%, agricultural volume increase is up to 23% or more.
Detailed description of the invention
Phyletic evolution tree graph constructed by Fig. 1: JSCX-1 genome and the higher strain sequence of homology.
Specific embodiment
It will be specifically described, but be not to be construed as to the present invention by the way that specific embodiment is further to the present invention below The restriction of protection scope.
Embodiment 1
The identification of the physiological and biochemical test and 16S rDNA molecule of bacterial strain
As shown in Figure 1, Bacillus strain JSCX-1 carries out activation culture at being 29-31 DEG C in temperature on NA plate, NA culture medium: beef extract (Beef Extract) 3g, peptone (Peptone) 10g, sodium chloride (NaCl) 5g, agar powder (Agar) 15g, pH7.0.
It has been observed that the single colonie subcircular of JSCX-1, milky, long-term cultivation bacterium colony are inclined after incubation time is 20-24h Brown color, neat in edge is opaque, and fold is omited on surface.Bacterial strain JSCX-1 Gram's staining is purple, for the positive；Spore staining Middle gemma takes on a red color in green, thallus.Bacterial strain JSCX-1 is to grow along media surface in aerobic-type test result, shows bacterial strain JSCX-1 is aerobic bacteria.
Physiological and biochemical test is oxidase test, hydrogen peroxide test, gelatin liquefaction test, citrate test, H2S is produced The result of raw test is the positive；Phenylalanine lyase test, Starch Hydrolysis test, indole test, methyl red test, acetyl Carbinol methine test, nitrate reduction test, result is feminine gender in salt tolerance test.
Bacillus strain JSCX-1 can containing 2%, 5%, grow in the NA culture medium of 8%NaCl, containing 10%NaCl NA culture medium in can not grow, the sugared fermentation broth flavescence color and inverted in glucose, mannose ferment test Moral Han Shi tubule has gas generation.The morphological feature and physiological and biochemical property and common bacteria system identification handbook of bacterial strain JSCX-1 Middle bacillus (Bacillus sp.) is the most similar.
Using JSCX-1 genome DNA as template, PCR amplification 16SrDNA obtains the amplified production of length about 1.5Kb, this The report that most highly conserved and consistent length is 1.5Kb or so in all bacteriums with 16SrDNA coincide.Arrange sequencing As a result the total length that can obtain carrying out the 16SrDNA sequence of Phylogenetic Analysis is 1419bp, by sequencing result in the website NCBI Blast is searched in GenBank database, compared with listed 16SrDNA sequence carries out nucleotide homology, finds bacterial strain The 16SrDNA sequence of JSCX-1 and more bacillus category bacterium (Bacillus sp.) similarities are higher, downloading and gemma bar The higher sequence of 16SrDNA sequence homology of bacteria strain JSCX-1 chooses the highest building systematic evolution tree of 10 plants of similarities, It was found that bacterial strain JSCX-1 and Bacillus sp.Q2-S1 (JX994136.1) are in same branch, homology highest is greater than 99%, in conjunction with cultural character and physiological and biochemical property qualification result, Bacillus strain JSCX-1 is initially identified as gemma bar Campylobacter bacteria (Bacillus sp.)
The present invention is not easy the Bacillus strain for making soybean phytophthora generate resistance, prevention and treatment root rot free from environmental pollution And its gemma bacillus agent.Gemma bacillus agent belongs to biological agent, absolutely not changes brought by the use because of chemical agent The problems such as learning drug resistance, thus be conducive to the No-harmful apple orchard of soybean.Peasant can not have to or reduce other prevention and treatment soybean phytophthoras Pesticide dosage.Peasant is reduced to reduce expenses.Meanwhile the control efficiency of the gemma bacillus agent is up to 50% or more, also Increase production function, income can be increased for peasant.Gemma bacillus agent is 55-64% to the control efficiency of root rot, and agricultural increases It produces up to 23% or more.
Embodiment 2
Steps are as follows for soybean phytophthora zoospore suspension preparation method:
(1) by the soybean phytophthora bacterial strain of preservation, activated growth, activated growth time are 5-6d on V8 culture medium.
(2) tomato and lettuce are cleaned to dry and is blended with tissue pulper.It is removed slag with four layers of filtered through gauze.According to tomato juice It is mixed with the ratio of lettuce juice 1:2.
(3) CaCO is added in the ratio of 0.02% (w/v)3, add the vegetable juice culture that deionized water is configured to 50% Base.Temperature is 120-122 DEG C, sterilization time 29-31min.
(4) by the soybean phytophthora bacterial strain of activation on LBA culture medium, temperature is 24-26 DEG C, and dark condition is cultivated, Incubation time with punch (d=5mm) takes bacterium dish along colony edge after being 3-4d.
(5) 4 ferfas dish are placed in 14-16ml culture medium, 24-26 DEG C, dark condition is cultivated, incubation time 3- 4d。
(6) incline culture medium, and the aqua sterilisa that volume is 19-21ml is added, and interval time is that 12-14h changes primary sterilizing again Water.
(7) in the case where temperature is 24-26 DEG C, constant temperature illumination box is cultivated, incubation time 20-24h, generates travelling Sporangium.
(8) zoosporangium suspension is subjected to homogenate 1min in the case where revolving speed is 3000-3200r/min, with sterile water tune Section to concentration is about 1 × 104-1×105CFU/ml。
A kind of Bacillus strain of prevention and treatment root rot of the invention, the bacterial strain are bacillus (BaciLLus.sp)；The entitled bacillus JSCX-1 of preservation；It is common to be preserved in China Committee for Culture Collection of Microorganisms Microorganism center (CGMCC), preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s；Preservation date: 2015 01 The moon 07；Deposit number: CGMCC No.10284.
The single colonie of the Bacillus strain is round, milky, the inclined brown color of long-term cultivation bacterium colony, neat in edge, Opaque, fold is omited on surface.The Bacillus strain Gram's staining is purple, for the positive；Gemma is in green in spore staining Color, thallus take on a red color；Bacillus strain is to grow along media surface in aerobic-type test result, is aerobic bacteria.
The gemma bacillus agent of the prevention and treatment root rot of bacillus preparation of the present invention.Its active constituent is such as Under at least one of (a), (b), (c) and (d):
(a) fermentation culture medium of bacillus described in claim 1；
(b) bacillus described in claim 1；
(c) gemma of bacillus described in claim 1；
(d) fermentation supernatant of bacillus described in claim 1.
The preparation method of the gemma bacillus agent of prevention and treatment root rot of the present invention, includes the following steps:
It (1) is bacillus, Bacillus upper life in NB culture medium by the Bacillus strain JSCX-1 Long, temperature is 29-31 DEG C, vibrates and is cultivated in the case where revolving speed is 150-180r/min, incubation time 16-20h, then super Separately sampled survey OD600 value in net workbench is compared in continuous mode with not connecing the NB culture medium of bacterium；The NB culture Base is made of beef extract 2.9-3.1g, peptone 9.9-10.1g, sodium chloride 4.9-5.1g；PH is 6.8-7.0, with distillation Said components are settled to 1000ml by water, and sterilize 19-21min at being 120-121 DEG C in temperature.
(2) when Bacillus culture to OD600 value is 0.5-0.8, by Bacillus solution and distilled water It being added in NB culture medium according to the volume ratio of 1:100, temperature is 28-30 DEG C, revolving speed is shaken cultivation under 150-180r/min, Incubation time is 20-24h, is then centrifuged in the case where revolving speed is 6000-8000r/min, disengaging time 9-11min, Gemma bacillus agent is made.By the concentration dilution of gemma bacillus agent to 1 × 108-1×1010CFU/ml。
Application of the gemma bacillus agent of the present invention in prevention and treatment root rot.
The measurement of excised leaf control efficiency
It is spare that gemma bacillus agent is diluted to scheduled concentration；The microbial inoculum 2-3d pouring root can be used or be broadcast after emergence Seed soaking uses before kind.Healthy leaves of the same size are acquired on neat soybean plant strain from growing, are rinsed well under tap water, It is impregnated with 70% alcohol, soaking time 30s, with being dried in superclean bench after aseptic water washing 2min.By Soybean Leaves Piece impregnates 1min wherein, and taking-up is dried in superclean bench.1 μ L soybean phytophthora zoospore suspension is connect on blade, To connect NB culture medium on blade as control, each processing is repeated 5 times.At 24-26 DEG C, relative humidity 80-90%, dark condition culture Lesion diameter is measured after 3-4d, assesses preventive effect.The calculation formula of preventive effect is: preventive effect (%)=(control group lesion diameter-processing group Lesion diameter)/control group lesion diameter × 100%.
The gemma bacillus agent that table 1 is bacillus JSCX-1 measures the control efficiency of soybean excised leaf.
Table 1
Such as table 1, it can be concluded that, soybean excised leaf is the experimental results showed that concentration is 108-10The JSCX-1 gemma bar of CFU/ml Bacteria agent has good inhibiting effect to inoculation soybean phytophthora zoospore on soybean leaves.JSCX-1 bacillus bacterium after 3-4d Agent is to the preventive effect of soybean phytophthora zoospore up to 64.21%.Excised leaf the experimental results showed that bacillus JSCX-1 gemma Bacillus microbial inoculum is 1 × 108-1×1010The concentration of CFU/ml has good inhibiting effect to soybean phytophthora.Different letter in table It indicates in 0.05 horizontal upper significant difference.
Embodiment 3
The measurement of potting control efficiency
Embodiment 3 the difference from example 2 is that: fill to the soybean seedling root for growing 2 true leaves such as bacillus bacterium Gemma bacillus agent, every plant of 15 ± 1ml of filling.The seedling of isometric NB culture medium is poured into root position as control.After 3-4d Soybean phytophthora zoospore suspension is filled to soybean seedling root, every plant pours into 30 ± 1ml.Each processing is repeated 5 times.In 24-26 DEG C culture, disease incidence, disease index and control efficiency are calculated after 9-11d.Each parameter calculation formula is as follows:
Disease incidence (%)=(disease plant number/total plant number) × 100%
Disease index=100 × ∑ (severity plant number × typical value at different levels at different levels)/(investigation plant number × superlative degree generation Tabular value)
Control efficiency (%)=[(control group disease index-processing group disease index)/control group disease index] × 100%
Table 2 is that bacillus JSCX-1 gemma bacillus agent measures the potting control efficiency of soybean blight；
Table 2
Such as table 2 it can be concluded that, inoculation soybean phytophthora zoospore liquid 9-11 days after detect, potted plant experiment the result shows that through The gemma bacillus agent leaching root processing of bacillus JSCX-1 is crossed, soybean plant strain can effectively resist the infection of soybean blight, preventive effect Up to 54.59%；Different letters is indicated in 0.05 horizontal upper significant difference in table.
Embodiment 4
To the growth-promoting effect measuring of soybean seedling
Embodiment 4 the difference from example 2 is that: pour into 15 ± 1ml gemma to the soybean seedling root for growing 4 leaves Bacillus bacterium gemma bacillus agent pours into the seedling of isometric NB culture medium using root as control, and each processing is repeated 3 times.15 Fresh weight, dry weight, root long and the stem length of big bean seedlings are measured after ± 1d, calculate biomass increment rate.The calculating of biomass increment rate is public Formula is: biomass increment rate (%)=(processing 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
Such as table 3, it can be concluded that, growth-promoting effect experiment shows the gemma bacillus agent root irrigation with bacillus JSCX-1 Big bean seedlings indices all increased after 15 ± 1d.Average every young plant fresh weight increases by 37.06%, and dry weight increases by 23.53%, Root long increase by 26.65%, stem length increase by 11.31%, Biological control the result shows that by bacillus JSCX-1 bacillus Big bean seedlings items growth indexes all increased after microbial inoculum root irrigation.Different letters is indicated in 0.05 horizontal upper difference in table Significantly；
Can prove that according to the above various embodiments is by the medicament of effective component of gemma bacillus agent of the present invention A kind of novel agricultural bacteriocide.This medicament is especially and shows brilliant effect to root rot, and to environment dirt Contaminate small, the low feature of drug resistance.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, the present invention Claimed range is delineated by the appended claims, the specification and equivalents thereof from the appended claims.

Claims (7)

1. a kind of Bacillus strain for preventing and treating root rot, it is characterised in that: the bacterial strain is bacillus (BaciLLus.sp)；The entitled bacillus JSCX-1 of preservation；It is common to be preserved in China Committee for Culture Collection of Microorganisms Microorganism center (CGMCC), preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s；Preservation date: 01 month 2015 07 day；Deposit number: CGMCC No.10284.
2. the gemma bacillus agent of the prevention and treatment root rot of bacillus preparation according to claim 1.
3. the gemma bacillus agent of prevention and treatment root rot according to claim 2, active constituent be following (a), (b), at least one of (c) and (d):
(a) fermentation culture medium of bacillus described in claim 1；
(b) bacillus described in claim 1；
(c) gemma of bacillus described in claim 1；
(d) fermentation supernatant of bacillus described in claim 1.
4. the method for preparing the gemma bacillus agent of prevention and treatment root rot described in claim 2 or 3, it is characterised in that: packet Include following steps:
(1) the Bacillus strain JSCX-1 is seeded in NB culture medium upper growth, temperature is 29-31 DEG C, is in revolving speed Oscillation is cultivated under 150-180r/min, incubation time 16-20h, then separately sampled survey OD600 in superclean bench It is worth, is compared in continuous mode with not connecing the NB culture medium of bacterium；
(2) when Bacillus culture to OD600 value is 0.5-0.8, by Bacillus solution and distilled water according to The volume ratio of 1:100 is added in NB culture medium, and temperature is 28-30 DEG C, revolving speed is shaken cultivation 20-24h under 150-180r/min, Then 9-11min is centrifuged in the case where revolving speed is 6000-8000r/min, gemma bacillus agent is made.
5. the preparation method of gemma bacillus agent according to claim 4, it is characterised in that: in step (2), by gemma The concentration dilution of bacillus microbial inoculum is to 1 × 108-1×1010CFU/ml。
6. the preparation method of gemma bacillus agent according to claim 4, it is characterised in that: in step (1), the NB Culture medium is made of beef extract 2.9-3.1g, peptone 9.9-10.1g, sodium chloride 4.9-5.1g；PH is 6.8-7.0, with steaming Said components are settled to 1000ml by distilled water, and sterilize 19-21min at being 120-121 DEG C in temperature.
7. application of the gemma bacillus agent described in claim 2 or 3 in prevention and treatment root rot.
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