CN103421722A - Litchi endogenous bacillus subtilis strain and biological agent and application thereof - Google Patents
Litchi endogenous bacillus subtilis strain and biological agent and application thereof Download PDFInfo
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- CN103421722A CN103421722A CN2013104012709A CN201310401270A CN103421722A CN 103421722 A CN103421722 A CN 103421722A CN 2013104012709 A CN2013104012709 A CN 2013104012709A CN 201310401270 A CN201310401270 A CN 201310401270A CN 103421722 A CN103421722 A CN 103421722A
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Abstract
The invention belongs to the field of biopesticides and discloses a litchi endogenous bacillus subtilis strain and a biological agent and application thereof. The litchi endogenous bacillus subtilis strain is named bacillus subtilis NMB-8 and has been preserved in the China Center for Type Culture Collection (CCTCC) which is located in Wuhan University in Wuhan, China on Aug 19, 2013 and the preservation number is CCTCC No: M2013376. The bacterial strain is from litchi endogenous bacteria and can be well colonized on the surface of a litchi fruit. The litchi endogenous bacillus subtilis strain can prevent peronophythora litchi effectively and has a good peronophythora litchi biocontrol function. The biogenetic derivation of the litchi endogenous bacillus subtilis strain is environmentally friendly and non-toxic. The litchi endogenous bacillus subtilis strain can be sprayed onto the surface of the litchi fruit for disease control, influence on the environment is light, and safety and reliability of litchi fruits and products processed from the litchi fruits are guaranteed. Requirements for the culturing condition of the litchi endogenous bacillus subtilis strain are low, and the development and application prospect is wide.
Description
Technical field
The invention belongs to field of biological pesticide, particularly a strain lichee endophytic Bacillus subtilis and biotechnological formulation and application.
Background technology
Lichee (Litchi chinensis Sonn) is important subtropics cash crop, and fruit flavor is good, look beautiful, nutritious, have the laudatory title of " China really precious ", and its economic worth is high, is China's one of the most competitive fruit in the international market.Lichee, in harvesting, transportation and sales and storage, caused huge financial loss because rotting, and post-harvest diseases is to cause rotten major cause.Cause that the rotten Major Diseases of fruit rot has peronospora tabacina, anthrax, sour rot etc., the peronospora tabacina wherein caused by Peronophythora Litchii germ (Peronophythora litchi Chen ex Ko et al.) be the lichee postharvest storage fresh-keeping in topmost disease.Yet at present, in production, the control of this disease mainly be take to Agro-chemicals control as main.
Bacillus (Bacillus) is a kind of antagonistic bacterium of broad spectrum, has a plurality of genus bacillus subgroups to have consequence in biocontrol of plant disease.Yang Shaobo etc. have reported that the bio-active substance mass-energy that bacillus polymyxa (Paenibacillus polymyxa) produces has antagonistic action significantly to pathogenic bacterias such as Fusarium oxysporum (Fusarium oxysporum), aspergillus nigers (Aspergillus niger), Chen Haiying etc. screen a strain Paenibacillus polymyxa has bacteriostatic action preferably to fungies such as melon anthrax bacteria, Rhizoctonia solani Kuhns, and Yuan Jun etc. have reported that bacillus pumilus (Bacillus pumilus) has significant restraining effect to Potato Ring Rot.For subtilis (Bacillus subtilis), Fan Qing etc. have reported this bacterium, and to adopting, rear citrus fruit is blue or green, green mould has inhibition significantly, Kong Jian etc. have reported that this bacterium passes the bacteriostatic activity of sickle-like bacteria and ring rot of apple etc. to multiple soil such as cotton are withered, Wang Ya equality has been reported the Biocontrol Effect of this bacterium to fusarium graminearum, and Baker etc. have reported that this bacterium has Biocontrol Effect significantly to the bean rust.
Therefore, the natural resources that subtilis is important as a class, caused domestic and international investigator's extensive concern, has the potentiality of exploitation biological pesticide.
Summary of the invention
In order to overcome the shortcoming and deficiency of above-mentioned prior art control peronophythora litchi, the application of exploitation subtilis, it is the screening object that primary and foremost purpose of the present invention is to provide a strain to take microorganism in litchi fruits, what separation obtained has remarkable inhibiting lichee endophytic Bacillus subtilis to the Peronophythora Litchii germ, called after subtilis (Bacillus subtilis) NMB-8, for the biological control of peronophythora litchi lays the foundation; In addition, the Antagonistic Endophytic that the present invention screens from lichee, use it for the control of lichee disease, only show as the growth of certain microbe population between specific population or subdue, thereby do not change the classification of microbial population on lichee and kept the stability of population, in addition due to itself and lichee long-term co-existence, reciprocal symbiosis, a series of survival mechanism similar to it have been formed, it is sprayed application to the control of lichee surface for disease, more safe and reliable concerning the edible and converted products of lichee.
Another purpose of the present invention is to provide a kind of biotechnological formulation prepared based on above-mentioned lichee endophytic Bacillus subtilis.
Still a further object of the present invention is to provide the application of above-mentioned lichee endophytic Bacillus subtilis in the control peronophythora litchi.
Purpose of the present invention realizes by following proposal:
One strain lichee endophytic Bacillus subtilis, by its called after subtilis (Bacillus subtilis) NMB-8, be preserved in Chinese Typical Representative culture collection center, be called for short CCTCC, address: Wuhan, China Wuhan University, preserving number is CCTCC No:M2013376, and the preservation time is on August 19th, 2013.
Described lichee endophytic Bacillus subtilis is 30 ℃ of cultivation 24h on the LB substratum, and the bacterium colony surface irregularity is opaque, normal wrinkle mould, dirty white or the micro-yellow of forming.
A kind of biotechnological formulation, prepare based on above-mentioned lichee endophytic Bacillus subtilis.
Described biotechnological formulation is prepared through liquid fermenting by the lichee endophytic Bacillus subtilis, preferably comprises following steps: the lichee endophytic Bacillus subtilis is seeded in the LB nutrient solution and cultivates, can obtain biotechnological formulation.
The pH value of described LB nutrient solution is 7.0.
Described cultivation refers at 28～30 ℃, and shaking table concussion speed is to cultivate 24h under 180～200rpm.
The application of above-mentioned biotechnological formulation in the control peronophythora litchi.
The application of above-mentioned lichee endophytic Bacillus subtilis in the control peronophythora litchi.
The present invention has following advantage and beneficial effect with respect to prior art:
1, the subtilis that the present invention obtains derives from the lichee endogenetic bacteria, can on the litchi fruits surface, surely grow well; The Antagonistic Endophytic that the present invention screens from lichee in addition, use it for the control of lichee disease, only show as the growth of certain microbe population between specific population or subdue, thereby do not change the stability that the classification of microbial population on lichee affects population, due to itself and lichee long-term co-existence, reciprocal symbiosis, formed a series of survival mechanism similar to it in addition, it is sprayed application to the control of lichee surface for disease, more safe and reliable concerning the edible and converted products of lichee.
2, subtilis of the present invention has strong restraining effect to the Peronophythora Litchii germ, has the Biocontrol Effect of excellent control peronophythora litchi, and this biogenetic derivation environment-protecting asepsis, little to eco-environmental impact.
3, the subtilis that the present invention obtains, culture condition requires low, has good development prospect.
The accompanying drawing explanation
Fig. 1 is the single bacterium colony figure of subtilis NMB-8 on the LB substratum.
Fig. 2 is the restraining effect design sketch of subtilis NMB-8 to the Peronophythora Litchii germ, and wherein, A is the inhibition figure of subtilis to the Peronophythora Litchii germ, and B is Peronophythora Litchii germ normal growth colonial morphology.
Fig. 3 is the as a result figure of subtilis NMB-8 on the impact of Peronophythora Litchii germ mycelia, and wherein, A is Peronophythora Litchii germ normal bacteria silk, and B is Peronophythora Litchii germ deformity mycelia.
Fig. 4 is the inhibition figure that the different bacillus subtilis strains that are separated to are sprouted Peronophythora Litchii germ sporocyst.
Fig. 5 is subtilis NMB-8 and biotechnological formulation thereof the prevention effect figure to Peronophythora Litchii, wherein, A is the LB nutrient solution, B is the clear water contrast, C is the subtilis bacteria suspension, D is subtilis bacteria suspension+Peronophythora Litchii germ, and E is subtilis filtrate+Peronophythora Litchii germ, and F is the Peronophythora Litchii germ.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Embodiment 1: the separation of lichee endophytic Bacillus subtilis, purifying and preservation
(1) configuration of LB substratum: take Tryptones (Tryptone, Oxoid LTD LP0042, England) 10g, yeast extract (Yeast extract, Oxoid LTD LP0021, England) 5g, sodium-chlor (NaCl, Chemical Reagent Co., Ltd., Sinopharm Group, 10019318) 10g, add water 1000mL and stir, then add 15g agar, fully packing after heating for dissolving, store standby after 121 ℃ of sterilizing 20min.
(2) separation of lichee endophytic Bacillus subtilis, purifying:
For separating of the litchi fruits sample pick up from respectively Agricultural University Of South China's gardening institute lichee base, Shenzhen's kylin mountain villa Litchi Garden and Nong Ke orchard, Bao'an, Shenzhen, selected Litchi Varieties is glutinous rice wrapped in lotus leaves, cv. Feizixiao and osmanthus flavor three large main breeds, is divided into three groups.
The separation method of endogenetic bacteria is mainly with reference to (He Hong such as He Hong, Cai Xueqing, Guan Xiong, Hu Fangping. capsicum endophytic Bacillus subtilis (Bacillus subtilis) BS-2 and the research of BS-1 control pepper anthracnose. Plant Pathology, 2003, 33 (2): method 170-173.) also slightly makes an amendment, concrete operations are as follows: the fruit of three groups of health is rinsed well with tap water respectively, take fruit 34g after drying moisture, alcohol-pickled 2～the 3min that is 70% by percent by volume, use again 5% clorox surface sterilization 2～10min, finally use aseptic water washing 4 times, on aseptic filter paper, after suck dry moisture, sample is proceeded in aseptic mortar, after adding the 10mL sterilized water to pulverize, static placement 15min, respectively get 100 μ L and be coated with the LB flat board, each processes 3 repetitions, after 30 ℃ of cultivation 48～72h, observations.
Take rinsing liquid method of inspection (Schulz et al, 1993.) sterilisation effect of the tissue surface of testing, the rinsing liquid of getting the last rinsing pasteurization material of 0.2mL is coated on the LB solid plate and is cultivated, observing to cultivate has or not bacterium colony to grow, if aseptic, drop out existing, the testimonial material surface sterilization is thorough, otherwise separating resulting can not be used.
Separate and obtain 7 strain bacterial strains, the purifying obtained strains is carried out to the ITS sequential analysis, be defined as subtilis (Bacillus subtilis), respectively called after subtilis NYB-3, subtilis NFB-35, subtilis NFB-25, subtilis NFB-21, subtilis NMB-8, subtilis NFB-28, subtilis NOB-1.Described bacillus subtilis strain NMB-8 separates the glutinous rice wrapped in lotus leaves fruit from kylin mountain villa, Shenzhen Litchi Garden, its sequence is as follows: the Bacillus subtilis homology that is EU346662.1 with the number of logging in GenBank reaches 98%, confirm that its Classification And Nomenclature is subtilis (Bacillus subtilis), called after subtilis (Bacillus subtilis) NMB-8.Sequence fragment is as follows:
TGGGAGGGACTTGTTCGACTTCACCCCAATCATCTGTCCCACCTTCGGCGGCTGGCTCCATAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTACTTAGCGTGCTTTCTGGTTAGGTACCGTCAAGTGCCGCCCTATTTGAACGGCACTTGTTCTTGCCTTACACAGAGCTTTACGATCCGAAAACATCATACTCACGCGTCGTGCTCGTCAGACTTCGTCATTGCGGAAAATACCTAATGGACTGACCCTCGTAAGGATTCTGGACA。
Subtilis NMB-8 is 30 ℃ of cultivations on the LB substratum, cultivate 24 hours, and the bacterium colony surface irregularity is opaque, normal wrinkle mould, dirty white or the micro-yellow (see figure 1) of forming.
(3) preservation of subtilis NMB-8: this bacterial strain is preserved in to the Chinese Typical Representative culture collection center of Wuhan, China Wuhan University, preserving number CCTCC No:M2013376 on August 19th, 2013.
Embodiment 2: the face-off culture method is measured the activity of subtilis
(1) preparation of LB substratum is with embodiment 1; The preparation of LB liquid nutrient medium is not except adding agar, and other formulas with the LB solid medium are identical, by claim medicament be sub-packed in Erlenmeyer flask (every bottle of 100mL nutrient solution) after fully dissolving, jump a queue, wrap up, 121 ℃ of sterilizing 20min, cooling rear storage is standby.
(2) preparation of Radix Dauci Sativae substratum: take the 200g Radix Dauci Sativae, by the juice extractor 16 layers of filtered through gauze of rear use of fully squeezing the juice, filtrate is supplied moisture to 1000mL, add again 15g agar, fully after heating for dissolving, be sub-packed in Erlenmeyer flask (every bottle of 100mL nutrient solution), 121 ℃ of sterilizing 20min, cooling rear storage is standby.
(3) preparation of the single bacterium colony of subtilis: by the subtilis streak inoculation to activation culture 24h on the LB culture medium flat plate, the preservation of ruling of picking list bacterium colony.
(4) preparation of subtilis bacteria suspension (biotechnological formulation) and filtrate: get in the LB nutrient solution Erlenmeyer flask that the single bacterium colony access step of subtilis (1) that step (3) prepares prepares, be placed in 30 ℃, shaking table speed is to cultivate 24h under 200rpm, can obtain the subtilis bacteria suspension; The bacteria suspension of acquisition, in the centrifugal 10min of 12,000rpm, is got to supernatant liquor, then used the strainer filtering thalline of 0.2 μ m, obtain subtilis filtrate.
(5) preparation of the cultivation of Peronophythora Litchii germ and sporangia suspension thereof: the Peronophythora Litchii germ that inoculation is numbered Zhaoqing 10 is to after cultivating 7d on the Radix Dauci Sativae flat board, the punch tool that is 5mm with diameter is beaten the mycelium inoculation of getting colony edge to after being placed under 25 ℃ on fresh Radix Dauci Sativae flat board and cultivating 7d, in flat board, adds the aseptic water washing sporocyst to be mixed with the sporangia suspension of desired concn.
(6) determination of activity:
1. subtilis is measured the restraining effect of Peronophythora Litchii pathogen growth: the subtilis streak inoculation that step (3) is made is to the central authorities of Radix Dauci Sativae culture medium flat plate, at the bacterium cake apart from this bacterium cake 3cm place inoculation Peronophythora Litchii germ, take in experiment and do not inoculate subtilis as contrast, 3 repetitions, be placed in 25 ℃ of lower constant temperature culture, when the contrast colony diameter reaches 6cm, measure antibacterial bandwidth, observe the mycelia form of the Peronophythora Litchii germ of antibacterial belt edge and contrast, result as shown in Figures 2 and 3.
2. the inhibiting mensuration that subtilis sprouts Peronophythora Litchii germ sporocyst: embodiment 1 is separated to the different bacillus subtilis strains that obtain and be seeded in the LB liquid nutrient medium that step (1) prepares and shake bacterium, being mixed with concentration according to step (4) is 10
8The subtilis bacteria suspension of cfu/mL is 4 * 10 with the sporangia suspension concentration that step (5) makes respectively by bacteria suspension, filtrate
4The Peronophythora Litchii germ sporangia suspension equal-volume of individual/ml mixes, and mixes in contrast with sterilized water, LB and sporangia suspension equal-volume.Get mixed solution 100 μ L and drip on aseptic concave surface slide glass, 25 ℃ of moisturizings are cultivated, and after 4h, take out and calculate sporangial germination rate, the results are shown in Figure 4.
Inhibiting rate (%)=(contrast sporocyst germination rate-processing sporocyst germination rate)/contrast sporocyst germination rate * 100
Interpretation of result: from Fig. 2, Fig. 3 and Fig. 4, lichee endophytic Bacillus subtilis NMB-8 has obvious restraining effect to the growth of Peronophythora Litchii germ, and caused the deformity of Peronophythora Litchii germ mycelia, the inhibition difference of different strains to the sporocyst germination rate of Peronophythora Litchii germ in addition, the bacteria suspension of the bacterial strain NMB-8 that wherein the present invention obtains and filtrate thereof are all sprouted utmost point restraining effect are significantly arranged the sporocyst of Peronophythora Litchii germ, bacteriostasis rate is respectively 97.4% and 88.6%, higher than other bacillus subtilis strain.
Embodiment 3: lichee endophytic Bacillus subtilis NMB-8 prevents and treats peronophythora litchi
(1) preparation of LB substratum is with embodiment 1.The preparation of Radix Dauci Sativae substratum and LB liquid nutrient medium is with embodiment 2.
(2) preparation of the preparation of lichee endophytic Bacillus subtilis NMB-8 bacteria suspension, the cultivation of Peronophythora Litchii germ and sporangia suspension thereof is with embodiment 2.
(3) the healthy fruit of consistent, the of uniform size lichee of picking ripening degree, after rinsing well in flowing water, dry, soak about 5min in the subtilis biotechnological formulation of respectively litchi fruits being prepared in step (2) and filtered liquid thereof, be taken out to drop and no longer drip, put into the moisturizing of sterilized antistaling box and process.By after above-mentioned processing inoculation 1d, adopt the sporangial method (10 of spray inoculation Peronophythora Litchii
4Individual/mL), cultivate the incidence of routine observation fruit after inoculation in being placed in respectively 25 ℃ of room temperatures and 4 ℃ (storage temperature of lichee).Process, only connect the subtilis biotechnological formulation with clear water processing, LB nutrient solution and process and only connect the processing of Peronophythora Litchii germ sporangia suspension in contrast.30 of litchi fruits are respectively selected in each processing, and 3 repetitions are set.
The indoor inoculation test of carrying out under the condition of 25 ℃ of medial temperatures, relative humidity 80%, the contrast that is connected to the Peronophythora Litchii germ after inoculation 36h starts morbidity, and inoculation has the processing not morbidity temporarily of subtilis biotechnological formulation and subtilis filtrate; After 3d is processed in inoculation, the sickness rate of germ contrast is 97.3%, and the sickness rate that subtilis biotechnological formulation and filtrate thereof were processed is only 14.4～33.3%, and prevention effect reaches 51.6～96%.
The indoor inoculation test of carrying out under the condition of 4 ℃ of medial temperatures, relative humidity 80%, the experimental result after storage is cultivated 20d is as table 1 and Fig. 5.
The biocontrol effect (4 ℃) of table 1 subtilis to lychee storage phase peronospora tabacina
Annotate: CK is the clear water contrast; Adopt the Duncan duncan's new multiple range method to carry out variance analysis, in data, same letter means difference not remarkable (P=0.05) on the same group.
From table 1, sickness rate and the disease index of the litchi fruits of processing through subtilis biotechnological formulation and filtrate thereof are all obviously low than the contrast that only connects the Peronophythora Litchii germ, and this shows that subtilis biotechnological formulation and filtrate thereof adopts rear peronospora tabacina to lichee and all have comparatively obvious preventive and therapeutic effect.Simultaneously, the processing that only is connected to Biocontrol Bacillus subtilis biotechnological formulation or its filtrate does not cause the lichee morbidity, illustrates that this bacterial strain is not the pathogenic bacteria of lichee, uses bacterial strain after can be used as the adopting of control peronophythora litchi.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (8)
1. a strain lichee endophytic Bacillus subtilis, it is characterized in that, by its called after subtilis (Bacillus subtilis) NMB-8, be preserved in Chinese Typical Representative culture collection center, be called for short CCTCC, address: Wuhan, China Wuhan University, preserving number is CCTCC No:M2013376, the preservation time is on August 19th, 2013.
2. subtilis according to claim 1 is characterized in that: described lichee endophytic Bacillus subtilis on the LB substratum 30 ℃ cultivate 24h, the bacterium colony surface irregularity is opaque, normal wrinkle mould, dirty white or the micro-yellow of forming.
3. a biotechnological formulation, is characterized in that: based on lichee endophytic Bacillus subtilis claimed in claim 1, prepare.
4. biotechnological formulation according to claim 3, it is characterized in that: by the lichee endophytic Bacillus subtilis, through liquid fermenting, prepared, specifically comprise following steps: the lichee endophytic Bacillus subtilis is seeded in the LB nutrient solution and cultivates, can obtain biotechnological formulation.
5. biotechnological formulation according to claim 4, it is characterized in that: the pH value of described LB nutrient solution is 7.0.
6. biotechnological formulation according to claim 4 is characterized in that: described cultivation refers at 28～30 ℃, and shaking table concussion speed is to cultivate 24h under 180～200rpm.
7. the application in the control peronophythora litchi according to the described biotechnological formulation of claim 3～6 any one.
8. the application of lichee endophytic Bacillus subtilis claimed in claim 1 in the control peronophythora litchi.
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| CN108902600A (en) * | 2018-07-02 | 2018-11-30 | 东莞市农业科学研究中心 | Application of the raw lactic acid bacteria in preparation low-sugar healthy type fermented juice in lichee |
| CN114015592A (en) * | 2021-09-30 | 2022-02-08 | 海南大学 | Endophytic biocontrol strain HN075 for preventing and treating litchi frost blight and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773699A (en) * | 2014-01-24 | 2014-05-07 | 华南农业大学 | Penicillium purpurogenum as well as biological preparation and application thereof in controlling litchi downy blight |
| CN103773699B (en) * | 2014-01-24 | 2016-01-06 | 华南农业大学 | The application of one strain penicillium purpurogenum and biotechnological formulation and control peronophythora litchi |
| CN108902600A (en) * | 2018-07-02 | 2018-11-30 | 东莞市农业科学研究中心 | Application of the raw lactic acid bacteria in preparation low-sugar healthy type fermented juice in lichee |
| CN108902600B (en) * | 2018-07-02 | 2021-06-04 | 东莞市农业科学研究中心 | Application of litchi endogenous lactic acid bacteria in preparation of low-sugar healthy fermented fruit juice |
| CN114015592A (en) * | 2021-09-30 | 2022-02-08 | 海南大学 | Endophytic biocontrol strain HN075 for preventing and treating litchi frost blight and application thereof |
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