CN103773697A - paecilomyces lilacinus and application thereof - Google Patents
paecilomyces lilacinus and application thereof Download PDFInfo
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- CN103773697A CN103773697A CN201310644028.4A CN201310644028A CN103773697A CN 103773697 A CN103773697 A CN 103773697A CN 201310644028 A CN201310644028 A CN 201310644028A CN 103773697 A CN103773697 A CN 103773697A
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Abstract
The invention discloses a paecilomyces lilacinus with a function of promoting growth of crops such as tomatoes, as well as a preparation method and application of a microbial inoculum thereof. A strain Paecilomyces lilacinus (Thom) Samson CGMCC (China General Microbiological Culture Collection Center) No.7994 has a function of promoting the growth of the tomatoes and the like. Wheat bran and water are uniformly mixed at a ratio of 1:1, the proper microbial inoculum Paecilomyces lilacinus CGMCC No.7994 is subjected to enlarge cultivationto prepare a wheat bran microbial inoculum. Tomato seedlings with consistent growth vigour are collected, and the microbial inoculum is spread around roots of the tomato seedlings. The strain is the paecilomyces lilacinus, is capable of remarkably promoting the growth of tomatoes, tobacco and the like, stable in effect, and better in popularization and application prospect.
Description
Technical field
The invention belongs to biotechnology prevention and control field, be specifically related to the preparation method of a Paecilomyces lilacinus (Paecilomyces lilacinus (Thom) Samson CGMCC No.7994) and microbial inoculum thereof and the application in control insect pest of the plant thereof.
Background technology
Facts have proved for many years: Paecilomyces lilacinus is prevented and treated usefulness to multiple nematode, its host has root knot nematode, Cyst nematode, golden nematode, golden nematode, is the most promising biological prevention and control agent of control root knot nematode.Because this bacterium can adapt to different weather condition, line eggs is had to higher parasitic rate, can effectively control soil nematodes quantity, obviously reduce harm, be widely used in production practice.
In the culturing process of Paecilomyces varioti, particularly in special culture medium and submerged fermentation culturing process, this bacterium can produce abundant derivative, the similar indolylacetic acid product of the first, its the most significant physiological function promotes the growth of root system of plant and plant while being lower concentration, therefore execute bacterium at root system of plant and can not only obviously suppress nematode infection, and can promote the growth of plant nutrition organ, the Germination and growth of seed is also had to promoter action simultaneously.But this type of research is also very limited, is necessary to strengthen Paecilomyces lilacinus to the plant especially research of cash crop growth promoting function
Summary of the invention
The object of the present invention is to provide the preparation method of a strain Paecilomyces lilacinus.
Further, the object of the present invention is to provide the application of a strain Paecilomyces lilacinus.
For achieving the above object, technical scheme of the present invention has adopted a strain Paecilomyces lilacinus, Paecilomyces lilacinus, and its preserving number is CGMCC NO.7994.Strain differentiation feature is as follows:
Molecular systematics is identified:
DNA extraction method: adopt quartzite sand grind CTAB extraction method (Scott et a1., 2000).
Genetic marker: rDNA ITS1-5.8S-ITS2, amplimer: ITS5, ITS4 (White et a1., 1990).
Pcr amplification reaction condition: first 94 degrees Celsius of heating make template DNA sex change for 3 minutes, then enter following temperature cycle: 94 degrees Celsius of sex change 30 seconds, anneal 30 seconds for 50 degrees Celsius, 72 degrees Celsius are extended 45 seconds, carry out altogether 35 circulations, finally extend 5 minutes (Wang, 2012) at 72 degrees Celsius.
PCR product detects and order-checking: PCR product and 100bp DNA ladder (MBI Fermentas) sepharose (agarose gel) with 2.0% in O.5 × TBE electrophoretic buffer under 80V voltage electrophoresis 20 minutes, then be placed in the ethidium bromide (EB of 0.5 μ g/ml, ehidium bromide) dye 15 minutes in solution, under the UV-light of wavelength 365 and 254nm, detect product for obvious single band and carry out two-way straight-through order-checking with ABI3700 (Applied Biosystems) after by Qing Ke Bioisystech Co., Ltd purifying.
Data processing and statistical analysis: original series is with obtaining sequence accurately after biosoftware Bioedit7.0.9 (Hall, 1999) editor, and the pattern of downloading with GenBank becomes sequence data matrix with authoritative bacterial strain sequence set.For sequence matrix, under Bioedit7.0.9, be necessary human-edited and adjust the new matrix obtaining.By this MEGA5.0 (Tamura et a1. for matrix, 2011) carry out neighbours' connection method (neighbor-joining, NJ) analyze, infer phylogenetic tree, and carry out the reliability of 1000 each branches of repeat assessment by the method for bootstrapping (bootstrap).
Bacterial strain TRCC23001rDNA ITS1-5.8S-ITS2 sequence following (seeing accompanying drawing 1):
5’-TCTCCGTTGGTGAACCAGCGGAGGGATCATTACCGAGTTATACAACTCCCAAACCCACTGTGAACCTTACCTCAGTTGCCTCGGCGGGAACGCCCCGGCCGCCTGCCCCCGCGCCGGCGCCGGACCCGCGCCCGCCGCAGGGACCCCAAACTCTCTTGCATTACGCCCAGCGGGCGGAATTTCTTCTCTGAGTTGCACAAGCAAAAACAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCAGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGCATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCTCAGCCCCCCCGGGGGCCTCGGTGTTGGGGGACGGCACACCAGCCGCCCCCGAAATGCAGTGGCGACCCCGCCGCAGCCTCCCCTGCGTAGTAGCACACACCTCGCACCGGAGCGCGGAGGCGGTCCGCGTAAAACGCCCAACTTTCTTAGAGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAA-3’
Phylogenetic tree is shown in accompanying drawing 2.
Simultaneously, the present invention has adopted a kind of preparation method of Paecilomyces lilacinus bacteria agent, and wheat bran mixes in 1:1 ratio with water, gets and is placed in right amount 500 milliliters of triangular flasks, add a cover (121 degrees Celsius of rear moist heat sterilizations, 20 minutes), after bran mass is chilled to room temperature, access Paecilomyces lilacinus spore suspension, be placed in mould constant incubator (28 degrees Celsius), after cultivating 3d, mycelia is covered with substratum, substantially grows to 6d spore, can take out stand-by.
The described concrete composition of substratum consist of 1.5 grams, ammonium nitrate, 1 gram of Sodium phosphate dibasic, magnesium sulfate (MgSO
47H
2o) 0.5 gram, 0.5 gram, Repone K, 0.01 gram, ferrous sulfate, 30 grams of sucrose, distilled water 1000 milliliters.
Further, technical scheme of the present invention has adopted the application of Paecilomyces lilacinus to control tomato root-knot eelworm and promotion tomato growth.
The present invention tests according to ordinary method Paecilomyces lilacinus to the promoter action of tomato growth: prepares Paecilomyces lilacinus microbial inoculum, to tomato seeds pre-treatment, selects seed full and of the same size and grow seedlings in incubated at room temperature,
Choose the tomato seedling that growing way is consistent (3～4 leaves) and be placed in basin, 1 tomato seedling of every basin sprinkles microbial inoculum around root, does 9 repetitions, periodic measurement plant overground part height.
The Paecilomyces lilacinus the present invention relates to is obviously better than not adding the control group of Paecilomyces lilacinus to the somatotrophic effect of tomato.
Control group experiment arranges: do not connect bran mass and the clear water processing of bacterium.
The mensuration of growth-promoting effect: in pot experiment process, except keeping the skin wet in time, measure and record the height of plant overground part every 3d.
Every the Accumulation biomass increment of the each treatment group tomato plant of 3d overground part, the tomato plant strain growth speed of Paecilomyces lilacinus wheat bran microbial inoculum processing is fast, 6d starts, its speed of growth is obviously faster than wheat bran substratum and clear water treatment group, increment every 3d exceedes 1cm, and the increment of the 5th 3d has reached 2.2cm, the poor growth of tomato seedling of wheat bran substratum and clear water control treatment, every 3d increment all, below 1cm, also only has 0.6cm when wherein growth is the fastest.
Bacterial strain of the present invention: Paecilomyces lilacinus (Paecilomyces lilacinus) has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), depositary institution's better address is: China, Beijing, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preservation date is: on 08 13rd, 2013, its preserving number was CGMCC NO.7994.
Embodiment 1 Paecilomyces lilacinus wheat bran microbial inoculum is to tomato growth promoting function
Wheat bran mixes in 1:1 ratio with water, get and be placed in right amount 500 milliliters of triangular flasks, add a cover (121 degrees Celsius of rear moist heat sterilizations, 20 minutes), after bran mass is chilled to room temperature, access Paecilomyces lilacinus spore suspension, be placed in mould constant incubator (28 degrees Celsius), after cultivating 3d, mycelia is covered with substratum, substantially grows to 6d spore, takes out stand-by.
The effect of embodiment 2 Paecilomyces lilacinus wheat bran microbial inoculums
Tomato seeds processing: adopt the method degerming of hoting water treatment of seeds, soak in cold water by tomato seeds 10 minutes, then put into the hot water of 50 degrees Celsius, constantly stir rapidly, seed is heated evenly, and supplement hot water at any time, by temperature instrumentation water temperature, make water temperature stability at 50～52 degrees Celsius, after 20～30 minutes, pull out and be placed on the waste heat that leaves in cold water, then soak seed in the warm water of 25～30 degrees Celsius 4～6 hours.
Grow seedlings: select fullly, the approaching tomato seeds of size, puts into seedling culture hole plate, 1, every cave seed, under room temperature, moisturizing is cultivated.
Potted plant with soil processing: in 120 degrees Celsius of baking ovens, to dry 4h, leave standstill to room temperature, break soil into pieces, add 6g composite fertilizer by every kg soil, mix, pack the flowerpot of 15 × 20cm into.
Test arranges: choose the tomato seedling that growing way is consistent (3～4 leaves) and be placed in basin, 1 tomato seedling of every basin, around root, sprinkle microbial inoculum, Paecilomyces lilacinus wheat bran microbial inoculum, the bran mass that does not connect bacterium and three groups of processing of clear water are established in test, process 9 repetitions, periodic measurement plant overground part height for every group.
In pot experiment process, except keeping the skin wet in time, measure and record the height of plant overground part every 3d.
Finally it should be noted that, above example is only illustrative rather than definitive thereof technical scheme of the present invention, although have been described in detail with reference to above-described embodiment, those of ordinary skill in the art is to be understood that: still can the present invention be modified or be replaced on an equal basis, the application of expansion bacterial classification of the present invention, and replace any modification and the part that do not depart from the spirit and scope of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
The growth promoting function of embodiment 3 Paecilomyces lilacinus microbial inoculums to other plant
Prepare Paecilomyces lilacinus agent method as above-mentioned embodiment 1, as follows to the experimental result of following plant:
| Crop | Tobacco | Tealeaves | Chinese cabbage | Eggplant | Cucumber | Strawberry | Oranges and tangerines | Persimmon | Rape |
| Volume increase (%) | 14.6 | 13.3 | 10.7 | 11.9 | 10.1 | 11.7 | 13.9 | 12.2 | 11.1 |
The growth-promoting effect of table 1 Paecilomyces lilacinus microbial inoculum
Note: Accumulation biomass increment=increment mean value ± SE, letter is thereafter to carry out the significance of difference after the new multipole difference analysis of Duncan, p < 0.05 with Dps7.05.
Accompanying drawing explanation
Fig. 1 is the 16S rDNA sequence of Paecilomyces lilacinus (Paecilomyces lilacinus (Thom) Samson CGMCC No.7994).
Fig. 2 is the phylogenetic tree of Paecilomyces lilacinus (Paecilomyces lilacinus (Thom) Samson CGMCC No.7994) based on 16sRNA sequence construct.
Claims (4)
1. a Paecilomyces lilacinus, Paecilomyces lilacinus (Thom) Samson, its preserving number is CGMCC NO.7994.
2. the preparation method of Paecilomyces lilacinus microbial inoculum, it is characterized in that: wheat bran and water in mass ratio 1:1 ratio mix, get and be placed in right amount 500 milliliters of triangular flasks, after adding a cover 121 degrees Celsius of moist heat sterilizations 20 minutes, after bran mass is chilled to room temperature, access Paecilomyces lilacinus spore suspension, is placed in mould constant incubator, cultivate mycelia after 3 days for 28 degrees Celsius and be covered with substratum, to the 6th day spore substantially grow take out after good stand-by.
3. the preparation method of Paecilomyces lilacinus microbial inoculum according to claim 2, is characterized in that: the described concrete composition of substratum consist of 1.5 grams, ammonium nitrate, 1 gram of Sodium phosphate dibasic, magnesium sulfate (MgSO
47H
2o) 0.5 gram, 0.5 gram, Repone K, 0.01 gram, ferrous sulfate, 30 grams of sucrose, 1000 milliliters of distilled water.
4. the application in promotion tomato growth according to Paecilomyces lilacinus described in claim 1-3.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104745483A (en) * | 2015-02-05 | 2015-07-01 | 山东蓬勃生物科技有限公司 | Paecilomyces variotii bacterial strain SJ1 and application thereof |
| CN105349434A (en) * | 2015-12-01 | 2016-02-24 | 北京市农林科学院 | Penicillium purpurogenum for poisoning plant parasitic nematodes and preparing method and application thereof |
| CN105602854A (en) * | 2014-11-24 | 2016-05-25 | 勐海茶业有限责任公司 | Bacterial strain of Paecilomyces lilacinus and application thereof to production of Pu'er tea |
| CN105779302A (en) * | 2016-03-24 | 2016-07-20 | 江西天人生态股份有限公司 | Hypocrea virens-containing composite microbial agent for controlling cucumber fusarium wilt, and preparation method of hypocrea virens-containing composite microbial agent |
| CN105901017A (en) * | 2016-05-24 | 2016-08-31 | 江西天人生态股份有限公司 | Agricultural nematicidal composition and application thereof |
| CN106376602A (en) * | 2016-08-31 | 2017-02-08 | 江西天人生态股份有限公司 | Paecilomyces lilacinus granule and preparation method thereof |
| CN106417387A (en) * | 2016-09-23 | 2017-02-22 | 江西天祥通用航空股份有限公司 | Paecilomyces lilacinus oil suspension agent and preparation method |
| CN106489929A (en) * | 2016-08-31 | 2017-03-15 | 江西天人生态股份有限公司 | A kind of exhibition film oil-suspending agent and its application in terms of preventing and treating crops sucking pest |
| CN106967613A (en) * | 2017-02-24 | 2017-07-21 | 河南省农业科学院园艺研究所 | Have pale purple purple spore bacteria strain and its application of High pathogenicity to botrytis cinerea |
| CN107058120A (en) * | 2017-02-24 | 2017-08-18 | 河南省农业科学院园艺研究所 | Have pale purple purple spore bacterium and its application of High pathogenicity to cucumber root-knot nematode |
| CN107446827A (en) * | 2017-09-15 | 2017-12-08 | 广州市博仕奥生物科技有限公司 | One plant of pale purple purple spore bacterium and its application |
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| CN104745483B (en) * | 2015-02-05 | 2017-07-11 | 山东蓬勃生物科技有限公司 | A kind of Paecilonyces variotii strain SJ1 and its application |
| CN104745483A (en) * | 2015-02-05 | 2015-07-01 | 山东蓬勃生物科技有限公司 | Paecilomyces variotii bacterial strain SJ1 and application thereof |
| CN105349434B (en) * | 2015-12-01 | 2019-01-15 | 北京市农林科学院 | The penicillium purpurogenum and its preparation method and application of one plant of intoxicating plant parasitical eelworm |
| CN105349434A (en) * | 2015-12-01 | 2016-02-24 | 北京市农林科学院 | Penicillium purpurogenum for poisoning plant parasitic nematodes and preparing method and application thereof |
| CN105779302A (en) * | 2016-03-24 | 2016-07-20 | 江西天人生态股份有限公司 | Hypocrea virens-containing composite microbial agent for controlling cucumber fusarium wilt, and preparation method of hypocrea virens-containing composite microbial agent |
| CN105901017A (en) * | 2016-05-24 | 2016-08-31 | 江西天人生态股份有限公司 | Agricultural nematicidal composition and application thereof |
| CN106376602A (en) * | 2016-08-31 | 2017-02-08 | 江西天人生态股份有限公司 | Paecilomyces lilacinus granule and preparation method thereof |
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| CN106417387A (en) * | 2016-09-23 | 2017-02-22 | 江西天祥通用航空股份有限公司 | Paecilomyces lilacinus oil suspension agent and preparation method |
| CN106967613A (en) * | 2017-02-24 | 2017-07-21 | 河南省农业科学院园艺研究所 | Have pale purple purple spore bacteria strain and its application of High pathogenicity to botrytis cinerea |
| CN107058120A (en) * | 2017-02-24 | 2017-08-18 | 河南省农业科学院园艺研究所 | Have pale purple purple spore bacterium and its application of High pathogenicity to cucumber root-knot nematode |
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| CN107446827B (en) * | 2017-09-15 | 2020-05-19 | 广州市博仕奥生物科技有限公司 | Purple lilac spore bacterium and application thereof |
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| CN111671136B (en) * | 2020-07-24 | 2022-04-15 | 四川中烟工业有限责任公司 | Method for solid state fermentation of tobacco stems and cut stems and application thereof |
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