CN101962623A

CN101962623A - Bacillus stain for preventing and controlling rice blast and rice sheath blight

Info

Publication number: CN101962623A
Application number: CN2010100281327A
Authority: CN
Inventors: 徐正君; 姜凯; 李利华; 高晓玲; 高鸿; 张小平; 马炳田; 徐永菊; 山口五十麿; 朱建清; 邓晓建; 钟凯; 王平荣; 黄静
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2010-01-19
Filing date: 2010-01-19
Publication date: 2011-02-02
Anticipated expiration: 2030-01-19
Also published as: CN101962623B

Abstract

The invention discloses Bacillus pumilux XZJ.BP01 having antagonistic effect on rice sheath blight and rice blast pathogenic bacteria, which is separated from osmanthus fruit. The strain was collected in China Center for Type Culture Collection on October 27th, 2009, with a collection number of CCTCC No.M209235. The strain XZJ.BP01 has antagonistic effect on the rice blast and rice sheath blight pathogenic bacteria, and the biological metabolic products of the strain has a sheath blight bacterium inhibition rate of over 99 percent; meanwhile, in field test of rice blast, the infection index of the rice applied with the fermentation solution of the strain is 16.7 percent, while the infection index of reference rice is up to 67.8 percent. As a result, the strain of the invention has a great significance for solving rice blast and rice sheath blight and has a remarkable effect.

Description

A kind of bacterial strain of bacillus of preventing and treating rice blast and banded sclerotial blight

Technical field

The present invention relates to the bacterial strain of a kind of bacillus Pseudomonas, especially a kind of from the sweet osmanthus fruit, separate obtain rice blast and banded sclerotial blight pathogenic bacteria are had the strain X ZJ.BP01 of bacillus pumilus (Bacilluspumilus) Pseudomonas of strong antagonistic action, the secondary metabolite that this bacterial strain produces in the PDA substratum, under 30 ℃ of culture condition also has extremely strong antagonistic action to banded sclerotial blight, the rice blast of paddy rice.

Background technology

Paddy rice is one of important crops.But along with rapid economic development, the progressively increase of population and the rapid minimizing of cultivated area add the attack of disease and pest, and Rice Production is being faced with the increasing pressure.Rice blast (Pyricularia) and banded sclerotial blight (Thanatephorus cucumeris) are two maximum big diseases of China rice district harm.Rice blast distributes very extensive, and all there is generation a countries and regions surplus in the of global 80, and the general popular time can be caused 10%～20% the underproduction, and its loss can reach 40%～50% when seriously taking place.Since the nineties, area takes place all more than 3,800,000 hectares in the year of China's rice blast, and annual loss paddy reaches several hundred million kilograms.Due to illness do harm to the different of generation period and position, rice blast promptly shows as seedling pest, leaf pest, symptoms such as joint pest, panicle blast and grain pest.The saprophytic ability of banded sclerotial blight pathogenic bacteria is strong, and host range is wide.Along with the popularization and the high fertile dense planting The Application of Technology of paddy rice fine quality, this evil of being critically ill is on the rise, and all can produce to paddy rice and cause certain loss every year, directly threatens the stable high yield of paddy rice.Therefore, people are in rice breeding seed selection process at present, and except considering rice yield and quality, the rice blast of rice varieties and sharp eyespot resistance also are used as an important breeding target.Up to the present in rice blast and sharp eyespot resistance screening breeding process,, also obtained certain achievement though people have paid a large amount of painstaking effort,, the result also is not very desirable.So far do not have also to find that rice blast and banded sclerotial blight are all had high anti-rice varieties.The physiological strain variation of rice blast is exceedingly fast, and a general resistant variety can only be kept 2-5 on producing, and this has brought very big difficulty to breeding; And banded sclerotial blight does not still have resistant variety at present in actual production, and people mainly prevent and treat banded sclerotial blight with jinggangmeisu, does not find other more satisfactory alternative microbiotic yet.On producing, the main tricyclazole class medicine that uses of rice blast control at present.Because the tricyclazole class belongs to chemical synthetic drug, effect is better in a short time though it is applied in, if long-term big area is used similar chemical agent continuously, can serious environment pollution, and can cause also that pathogenic bacteria is anti-, resistance increases considerably.If things go on like this, can be to the stability of whole agricultural, and human beings'health produces serious threat.Therefore, exploration discovery above pathogenic bacteria is had in good prevention effect, the physical environment can degrade, harmless antimicrobial substance, seem particularly urgent and important.

Summary of the invention

The object of the present invention is to provide a kind of bacillus pumilus XZJ.BP01 bacterial strain that obtains that from the sweet osmanthus fruit, separates.The secondary metabolite that this bacterial strain produces in substratum such as PDA, under the 28-30 ℃ culture condition has stronger preventive and therapeutic effect to rice sheath blight disease, rice blast.

Concrete technical scheme of the present invention is as follows:

Bacillus pumilus XZJ.BP01 bacterial strain among the present invention on October 27th, 2009 in that " Chinese typical culture collection " center " carries out preservation, and deposit number is: CCTCC NO:M 209235.

Strain X ZJ.BP01 of the present invention has following character:

1, morphological specificity:

On PDA or NA substratum, grow bacterium colony circle, neat in edge.The opticmicroscope result is behind the gramstaining: Gram-positive, tool gemma.Cultivated 3 days for 28 ℃ on the PDA substratum, the electron microscopic observation result is: atrichia, shaft-like, and size is 0.5-0.7 μ m * 1.1-2.4 μ m.

2, physiology, biochemical character (seeing the following form):

3, strain identification result

Aspect morphology, strain X ZJ.BP01 shows as bacillus pumilus.The bacillus pumilus physiological and biochemical property of part Physiology and biochemistry measurement result and standard is variant.For example this bacterial strain urobenzoic acid experiment is negative.Through 16s rRNA order-checking comparison, we find that strain X ZJ.BP01 and bacillus pumilus sequence similarity reach 99%, assert that it is the new bacterial strain of bacillus pumilus class.

Strain X ZJ.BP01 of the present invention all has antagonistic action to rice sheath blight disease and rice blast pathogen.The secondary metabolite of this strain X ZJ.BP01 has the strongly inhibited effect to the banded sclerotial blight growth of pathogenic bacteria simultaneously, and the bacteriostasis rate of banded sclerotial blight pathogenic bacteria is reached more than 99%.In field rice blast control test, be sprayed with the paddy rice of this strain X ZJ.BP01 secondary metabolite, its susceptible index is 16.7%, is 67.8% and contrast susceptible index.Illustrate that this strain X ZJ.BP01 secondary metabolite has good preventive effect to rice blast.

Description of drawings

Fig. 1 is the form (comprising A, B and C) of strain of i (bacillus) pumilus XZJ.BP01

Fig. 2 is the antagonistic action (comprise D and E) of strain of i (bacillus) pumilus XZJ.BP01 to rice blast and banded sclerotial blight pathogenic bacteria

Fig. 3 is the teratogenic effect (comprise F, G, H and I) of strain of i (bacillus) pumilus XZJ.BP01 to the rice sheath blight disease pathogenic bacteria

Fig. 4 suppresses rice sheath blight disease growth of pathogenic bacteria (comprising J and K) for the secondary metabolite of strain of i (bacillus) pumilus XZJ.BP01

Wherein, A is a strain X ZJ.BP01 colony morphology characteristic, B is strain X ZJ.BP01 gramstaining result, C is that strain X ZJ.BP01 electron microscopic morphology is observed, D is the antagonistic action of strain X ZJ.BP01 to the rice blast pathogenic bacteria, E is the antagonistic action of strain X ZJ.BP01 to the rice sheath blight disease pathogenic bacteria, F is the normal mycelia of Rhizoctonia solani Kuhn, G is that Rhizoctonia solani Kuhn mycelial growth generation spiral twines, and have and significantly walk quickly and keep away the antimicrobial direction of growth short of money, H is that phenomenon appears expanding in Rhizoctonia solani Kuhn mycelia tip, I is that cracking partly takes place the Rhizoctonia solani Kuhn mycelia, J is a sheath blight fungus growing state when not adding the secondary metabolite of strain X ZJ.BP01 in the substratum, and K is a sheath blight fungus growing state when having added the secondary metabolite of strain X ZJ.BP01 in the substratum.

Embodiment

Embodiment 1 strain X ZJ.BP01 screening method

70% ethanol carries out disinfection to the sweet osmanthus fruit, and aqua sterilisa cleans 3 times.Then fruit is pulverized centrifugally, obtained supernatant liquor, (1000 milliliters of potato 200 grams, sucrose 15 grams, agar 18 grams, water, pH7.0), inoculation rice blast and banded sclerotial blight are former, place 28 ℃ of constant incubators to cultivate to coat the PDA solid medium.Filter out the bacterial strain that suppresses the pathogenic bacteria growth.Isolate the mono-clonal bacterial strain through streak culture method.

Embodiment 2 antagonism bacterium antibiosis researchs

Inoculate Rhizoctonia solani Kuhn and strain X ZJ.BP01 in the plate that the PDA substratum is housed simultaneously, inoculation bacterium cake diameter is 1 centimetre.5 centimetres of two bacterium cakes distances.Inoculation is placed in 28 ℃ of constant incubators cultivates, and observes colonial morphology after 6 days.

With sheath blight fungus and strain X ZJ.BP01 face-off method inoculation, the sheath blight fungus filament at picking face-off edge and normal mycelium observe under the ordinary optical mirror and electron-microscope scanning detects.

The evaluation of embodiment 3 bacterial strains

A, morphological specificity: cultivate after 3 days for 28 ℃ on the PDA substratum, carry out conventional thalli morphology with scanning electronic microscope and opticmicroscope and observe.

B, Physiology and biochemistry experiment: with reference to the content of " uncle's Jie Shi Bacteria Identification handbook (the 8th edition) " bacterial strain is carried out Physiology and biochemistry and identify.

Employing bacterium universal primer P2F (5 '-agccaggatcaaactctg-3 '), P5R (5 '-aggaggtgatccagccgca-3 ') is to the 16rDNA of the strain X ZJ.BP01 order-checking of increasing, compare on NCBI with the 16rDNA that obtains, reach 99% with the bacillus pumilus similarity, it is bacillus pumilus for a primitive decision.

The experiment of embodiment 4 bacterial strain secondary metabolite antibiosis

Strain X ZJ.BP01 cultivates 24h in the PDB substratum, then that fermented liquid is centrifugal, filtration sterilization, get 50 ℃ of abundant mixings of PDA of 1ml and xx ml after, pour the sterilization culture dish of diameter 6cm into.After treating culture medium solidifying, the former bacterium cake of the banded sclerotial blight of 8mm diameter is connected to the substratum center of containing fermented liquid.Simultaneously, the former bacterium cake of banded sclerotial blight that connects the 8mm diameter be connected to contain PDB the substratum center in contrast.Contrast is except changing the 1ml fermented liquid into the pure PDB of 1ml, and other steps are all adopted and used the same method.Cultivate observations after 8 days down for 30 ℃.

During rice plant growth's to a leaf one core (about 10cm), on plant leaf, smear the fermented liquid of strain X ZJ.BP01, after 6 hours, spray the rice blast spore, adjoining tree changes the fermented liquid of strain X ZJ.BP01 into nonvaccinated liquid nutrient medium, and other condition is constant.Plant is incubated, preserves moisture with plastics film.Treat the susceptible situation of 1 week back detection.Handle and contrast and respectively get 50 strains, calculate susceptible index.

Claims

1. bacterial strain of bacillus of preventing and treating rice blast and banded sclerotial blight, it is characterized in that: this bacterial strain is bacillus pumilus (Bacillus pumilus) XZJ.BP01, and its deposit number is: CCTCCNO:M209235.

2. the new bacterial strain of bacillus of control rice blast according to claim 1 and banded sclerotial blight is characterized in that: this bacterial strain on the NA substratum 28 ℃ cultivated 24～36 hours, the opticmicroscope result is behind the gramstaining: Gram-positive, tool gemma.

3. the bacterial strain of bacillus of control rice blast according to claim 1 and banded sclerotial blight, it is characterized in that: this bacterial strain on the PDA substratum 28 ℃ cultivated 3 days, the electron microscopic observation result is: atrichia, shaft-like, and size is 0.5-0.7 μ m * 1.1-2.4 μ m.