CN103205372B - Paenibacillus alvei and its applications - Google Patents
Paenibacillus alvei and its applications Download PDFInfo
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- CN103205372B CN103205372B CN201210536839.8A CN201210536839A CN103205372B CN 103205372 B CN103205372 B CN 103205372B CN 201210536839 A CN201210536839 A CN 201210536839A CN 103205372 B CN103205372 B CN 103205372B
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- 241000178961 Paenibacillus alvei Species 0.000 title claims abstract description 52
- 241000223221 Fusarium oxysporum Species 0.000 claims abstract description 30
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- 241000596148 Crocus Species 0.000 claims description 14
- 230000008485 antagonism Effects 0.000 claims description 4
- 201000010099 disease Diseases 0.000 abstract description 17
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 17
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
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- 241000233866 Fungi Species 0.000 description 2
- 241000294743 Gamochaeta Species 0.000 description 2
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Abstract
The invention discloses a Paenibacillus alvei. The Paenibacillus alvei has a strain number of ZJUB2011-1 and has a preservation number of CCTCC M 2012448. The invention also discloses a microbial inoculum containing the Paenibacillus alvei. The invention further discloses applications of the Paenibacillus alvei in antagonistic Fusarium oxysporum and stigma croci bulb rot disease control. Compared with the prior art, the Paenibacillus alvei ZJUB2011-1 can efficiently inhibit the growth of Fusarium oxysporum, can be used for controlling plant diseases comprising the stigma croci bulb rot disease and the like caused by the invasion of Fusarium oxysporum, and has a wide application prospect.
Description
Technical field
The development and utilization field that the invention belongs to microorganism germ plasma resource, relates in particular to a kind of bacillus alvei and application thereof.
Background technology
Style and stigma of Saffron Crocus (Stigma Croci) brings considerable economic benefit can to plantation family as a kind of famous and precious medicinal material, style and stigma of Saffron Crocus has become one of the Major agro-industry in many counties (city), the whole nation at present, but taking place frequently of style and stigma of Saffron Crocus kind ball canker brought the loss can not be ignored to this industry in recent years.The killed posterior lobe jaundice of Crocus bulb, browning look, wilt witheredly, bulb blackout rots to stay ghost.According to investigations and statistics, rotten ratio occurs the general time Crocus bulb in Zhejiang Province is 5% left and right, and serious time part is planted family Crocus bulb and rotted to reach more than 40%, even all rots.
The pathogenic bacterium of the bulb of falling ill are carried out to isolation identification result and show, Crocus bulb canker is infecting and causing alternately by a kind of Fusarium oxysporum (Fusarium oxysporum) and a kind of anthrax-bacilus (Anthracnose sp.).Crocus bulb canker early stage is mainly that Fusarium oxysporum is infected and causes, and then anthrax-bacilus infects, and increases the weight of the state of an illness.
Crocus bulb canker has a strong impact on the quality of bulb and style and stigma of Saffron Crocus, and the Main Means of preventing and treating at present this disease is to use chemical bactericide.But because long-term a large amount of agricultural chemicals that use cause the problem of resistance enhancing, natural ecological environment pollution and pesticide residue more and more outstanding, the new way of seeking control of crop disease is the target that people make great efforts always.
Utilize biological control means to prevent and treat crop pest and can effectively avoid above-mentioned shortcoming, thereby there is wide development prospect.Wherein antagonistic bacterium, as the important biological control preparation of a class, occupies important one seat in the integrated control of Plant diseases.
The main advantage of bacterium in biological control application is: kind and the One's name is legion of (1) bacterium, exist in a large number at plant rhizosphere and overground part; (2) bacterium is wider to the mode of action of pathogenic bacteria, can be produced the modes such as resistance pathogenic bacteria is exerted an influence by competition, antagonism and parasitism, inducing plant; (3) there is surprising reproduction speed; (4) many bacteriums are present in plant rhizosphere and overground part, suitable to the ecology comparison of plant; (5) bacterium mostly can artificial culture, is convenient to control easy handling in practice; (6) some bacterium can not only prevent and treat disease and have growth-promoting functions, can increase crop yield.
Paenibacillus polymyxa (Paenibacillus polymyxa) is that series bacillus belongs to the type species of (Paenibacillus), it is the classificatory new development of bacillus that series bacillus belongs to, and within 1993, by Ash etc., 11 kinds is branched away from genus bacillus.The genus bacillus for biocontrol of plant disease with other kinds is similar, series bacillus also can produce multiple antimicrobial substance and produce gemma, there is very strong fecundity, some phytopathogens are had to obvious restraining effect, therefore can further develop biological pesticide.
Summary of the invention
The invention discloses a kind of bacillus alvei (Paenibacillus alvei) ZJUB2011-1, this bacterium can efficient antagonism Fusarium oxysporum (Fusarium oxysporum), can be used for control and is infected the Plant diseasess such as the Crocus bulb canker that causes by Fusarium oxysporum.
A kind of bacillus alvei, Classification And Nomenclature is bacillus alvei (Paenibacillus alvei), strain number is ZJUB2011-1, be preserved in the Chinese Typical Representative culture collection center (CCTCC) that is positioned at Luo Jia Shan, Wuhan City Wuhan University, preservation date is on November 6th, 2012, and preserving number is CCTCCM2012448.
This bacterial strain is Gram-positive facultative anaerobe.While growth on nutrient agar plate, without soluble pigment, bacterium colony circle, white, flat, smooth surface, moistening, neat in edge; In liquid medium within, leave standstill while cultivation, bacterium liquid muddiness, has throw out, and the bacterium liquid surface of being everlasting forms one deck mycoderm.The growth temperature of this bacterial strain is 15～45 ℃, and optimum growth temperature is 30～35 ℃; The most suitable growth pH is 7 left and right.
The microscopic morphology of bacillus alvei ZJUB2011-1 is observed to discovery, and the thalline of this bacterial strain is shaft-like, and size is 0.6～0.8 μ m × 2.2～4.5 μ m, without pod membrane.Gemma is oval, is positioned at thalline central authorities or sub-top, and gemma size is 0.7～0.9 μ m × 1.8～2.2 μ m.
Described bacillus alvei ZJUB2011-1 separates from style and stigma of Saffron Crocus epidermis, extracts the DNA of described bacillus alvei ZJUB2011-1, carries out pcr amplification and obtains its 16S rDNA sequence, and this 16S rDNA sequence is as shown in SEQ No.1.
The present invention also provides a kind of microbial inoculum that comprises described bacillus alvei.This microbial inoculum is by mixing bacillus alvei ZJUB2011-1 and suitable carrier.The concentration of described bacillus alvei ZJUB2011-1 is preferably 10
7～10
8cfu/mL.
The present invention also provides the application of described bacillus alvei in antagonism Fusarium oxysporum.Bacillus alvei ZJUB2011-1 of the present invention has more significant restraining effect to Fusarium oxysporum, and in an embodiment, inhibiting rate is 73%, higher than positive control subtilis (Bacillussubtilis) biocontrol microorganisms 1.
The present invention also provides the application of described bacillus alvei in control Crocus bulb canker, utilize the restraining effect of bacillus alvei ZJUB2011-1 to Fusarium oxysporum, bacillus alvei ZJUB2011-1 can be applied to control and be infected the Plant diseasess such as the Crocus bulb canker that causes by Fusarium oxysporum.
Compared with prior art, beneficial effect of the present invention is:
Bacillus alvei ZJUB2011-1 of the present invention can efficiently suppress the growth of Fusarium oxysporum, can be used for control and is infected the Plant diseasess such as the Crocus bulb canker that causes by Fusarium oxysporum, has a extensive future.
Accompanying drawing explanation
Fig. 1 is the gramstaining result figure of bacillus alvei (Paenibacillus alvei) ZJUB2011-1;
Fig. 2 is the transmission electron microscope observing figure of bacillus alvei (Paenibacillus alvei) ZJUB2011-1 nourishing body;
Fig. 3 is the growth conditions figure of bacillus alvei (Paenibacillus alvei) ZJUB2011-1 on nutrient agar plate;
Fig. 4 is the growth conditions figure in bacillus alvei (Paenibacillus alvei) ZJUB2011-1 liquid medium within;
Fig. 5 is the in vitro antagonistic action figure of biocontrol microorganisms to Fusarium oxysporum; Wherein, be for 1,2, No. 3 bacillus alvei ZJUB2011-1, be for No. 4 subtilis 1,5 is not for delivering a child fungi-proofing negative control.
Embodiment
One, pathogenic fungi separation and purification
Gathered the healthy morbidity kind ball of planting ball and suffering from Crocus bulb canker of style and stigma of Saffron Crocus in indoor cultivation stage on the spot from yellow mud mountain, San Dou Zhen He village, Jiande City, Zhejiang Province.According to Koch rule, adopt tissue surface partition method, obtain this sick pathogenic fungi from the rotten position separation and purification of style and stigma of Saffron Crocus kind ball---Fusarium oxysporum (Fusarium oxysporum).
Two, Selection of Antagonistic Bacteria
Cut 5g style and stigma of Saffron Crocus epidermis and put into 45mL sterilized water and shake 20min, then therefrom draw 1mL and put to filling fully vibration in 9mL sterilized water test tube with ground stopper, make 10
-1diluent; By that analogy, be made for 10
-2, 10
-310
-4, 10
-5, 10
-6dilution; Get respectively 0.1mL10
-4, 10
-5, 10
-6the diluent coated plate (NA substratum) of concentration is cultivated 48h under 30 ℃ of conditions; Then the different single bacterium colony of picking cultural characteristic carries out streak culture until obtain pure culture, for subsequent use.
To separate the Fusarium oxysporum obtaining as indicator from style and stigma of Saffron Crocus morbidity kind of a ball in step 1, adopt dull and stereotyped face-off method, from the pure culture obtaining, screening is to the inhibited bacterial strain of Fusarium oxysporum.
After picking purifying, eugonic Fusarium oxysporum mycelia piece (diameter 7mm) is placed in the dull and stereotyped central authorities of PDA, the pure culture bacterial strain point being separated to is connected on to plate edge apart from 25mm place, center, each culture dish connects 4 points, cultivates 3d for 28 ℃, and acquisition one strain has the bacterial strain of inhibition to Fusarium oxysporum.
Three, biocontrol strain is identified
The form and the biological property that in step 2, separate the style and stigma of Saffron Crocus kind ball canker biocontrol bacteria obtaining are identified.
As shown in Figure 1, this bacterial strain is Gram-positive facultative anaerobe; Electronic Speculum microscopic morphology is observed and is found, the thalline of this bacterial strain is rod-short, and size is 0.6～0.8 μ m × 2.2～4.5 μ m(Fig. 2), without pod membrane.Gemma is oval, is positioned at thalline central authorities or sub-top, and gemma size is 0.7～0.9 μ m × 1.8～2.2 μ m.
When this bacterial strain is grown on nutrient agar plate, bacterium colony circle, white, flat, smooth surface, moistening, neat in edge (Fig. 3); In liquid medium within, leave standstill while cultivation, bacterium liquid muddiness, has throw out, and the bacterium liquid surface of being everlasting forms one deck mycoderm (Fig. 4).The growth temperature of this bacterial strain is 15～45 ℃, and optimum growth temperature is 30～35 ℃; The most suitable growth pH is 7.0 left and right.
Extract this strain gene group DNA; Take genomic dna as template, carry out pcr amplification and obtain 16S rDNA sequence, and Genbank on NCBI carries out sequence alignment, in conjunction with above-mentioned observable physiological and biochemical property, be bacillus alvei (Paenibacillusalvei) by this identification of strains, be numbered bacillus alvei ZJUB2011-1.
Four, biocontrol microorganisms Antifungal Activity in Vitro is measured
Beat and get eugonic Fusarium oxysporum bacterium cake with the punch tool that punching diameter is 7mm, transfer in the dull and stereotyped central authorities of PDA, at the bacillus alvei ZJUB2011-1 that connects respectively screening gained with it on four angles of 2cm, culture dish is placed in to 28 ℃ of thermostat containers and cultivates 6d, with the positive contrast of subtilis (Bacillus subtilis) biocontrol microorganisms 1 bacterium, do not connect the negative contrast of Antagonistic Fungi, each processing repeats 3 times.Suppress result as shown in Figure 5.
Adopt DPS software to carry out statistical study to each group of result.Analytical results demonstration, bacillus alvei ZJUB2011-1 has to Fusarium oxysporum the ability that suppresses more significantly, and inhibiting rate reaches 73.00%, higher than positive control bacterium.Experimental result is as shown in table 1 below.
The in vitro fungistatic effect of table 1. bacillus alvei ZJUB2011-1
| Process | Colony diameter (mm) | Inhibiting rate (%) |
| Bacillus alvei ZJUB2011-1 | 27.2±0.93 c | 73.00 |
| |
32.3±1.16 b | 66.18 |
| Do not connect Antagonistic Fungi | 81.7±0.89 a | 0 |
Note: the 1. multiple variance analysis result of digital upper right corner letter representation Deng Kenshi in table, the different letter representation significant differences of same column (P<0.05);
2. inhibiting rate (%)=100 × (negative control colony diameter-processing colony diameter)/(negative control colony diameter-7mm).
Five, biocontrol microorganisms live body protection effect is measured
Bacillus alvei ZJUB2011-1 is made to the bacteria suspension that concentration is 1 × 108CFU/mL, choose anosis worm harm, immerse in bacteria suspension without wound, ripening degree and style and stigma of Saffron Crocus kind ball of the same size, soak time is 30min, and on style and stigma of Saffron Crocus kind ball inoculate Fusarium oxysporum after 12h interval time.
Control group is set simultaneously, and control group comprises three contrasts: with the positive contrast of inoculation Fusarium oxysporum 1(chemical pesticide after 300 times of diluent seed soaking of conventional pesticide 50% carbendazol wettable powder); Take concentration as 1 × 10
8the positive contrast of inoculation Fusarium oxysporum 2(biological pesticide after subtilis (Bacillus subtilis) the biocontrol microorganisms 1 suspension seed soaking of CFU/mL); After sterilized water seed soaking, inoculate Fusarium oxysporum as blank.Put interior cultivation of incubator of 28 ℃, 10d " Invest, Then Investigate " incidence.Each processing arranges 3 repetitions, averages.Result is as shown in table 2.
The live body prevention effect of the each treatment solution of table 2. to Crocus bulb canker
| Process | Disease index | Prevention effect (%) |
| Bacillus alvei ZJUB2011-1 | 40.00 a | 57.14 |
| 50% derosal | 43.33 ab | 53.57 |
| Genus bacillus 1 | 45.33 ab | 51.43 |
| Sterilized water | 93.33 c | 0 |
The severity Scaling standard of Crocus bulb canker is:
(1) 0 grade:: asymptomatic;
(2) 1 grades: stem-bulb rot area accounts for 0～1/5 of whole bulb;
(3) 2 grades: stem-bulb rot area accounts for 1/5～2/5 of whole bulb;
(4) 3 grades: stem-bulb rot area accounts for 2/5～3/5 of whole bulb;
(5) 4 grades: stem-bulb rot area accounts for 3/5～4/5 of whole bulb;
(6) 5 grades: bulb is completely rotten.
Disease index=(class value of ∑ sick sheet number × state of an illness at different levels)/(investigating total sheet number × highest level value) × 100;
Prevention effect (%)=(negative control disease index-processing disease index)/negative control disease index × 100%.
In sum, bacillus alvei of the present invention (Paenibacillus alvei) ZJUB2011-1 has stronger restraining effect to causing the rotten Fusarium oxysporum of style and stigma of Saffron Crocus kind ball, and in live body diseases prevention test, its bacteria suspension has good prevention effect to style and stigma of Saffron Crocus kind ball canker, has good production application prospect.
Claims (5)
1. a bacillus alvei, is characterized in that, called after bacillus alvei (Paenibacillus alvei) ZJUB2011-1, and preserving number is CCTCC NO:M2012448.
2. a microbial inoculum that comprises bacillus alvei described in claim 1.
3. microbial inoculum as claimed in claim 2, is characterized in that, the concentration of bacillus alvei is 10
7～10
8cfu/mL.
4. the application of bacillus alvei as claimed in claim 1 in antagonism Fusarium oxysporum (Fusarium oxysporum).
5. the application of bacillus alvei as claimed in claim 1 in control Crocus bulb canker.
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| CN109892340B (en) * | 2019-02-28 | 2024-03-08 | 金华市农业科学研究院 | Application of paenibacillus nidae in preventing and treating potato scab |
| CN114134068A (en) * | 2021-10-26 | 2022-03-04 | 湖州市中心医院 | Paenibacillus polymyxa microbial agent, preparation method and application thereof |
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| CN108179125A (en) * | 2018-01-26 | 2018-06-19 | 中国科学院南京土壤研究所 | Red bacillus H7 and its application |
| CN108179125B (en) * | 2018-01-26 | 2020-09-22 | 中国科学院南京土壤研究所 | Bacillus gibsonii H7 and application thereof |
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