CN103789211B - One strain has the abyssal sediment source Paecilomyces lilacinus A65 of anti-tealeaves pathomycete activity - Google Patents
One strain has the abyssal sediment source Paecilomyces lilacinus A65 of anti-tealeaves pathomycete activity Download PDFInfo
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- CN103789211B CN103789211B CN201210432272.XA CN201210432272A CN103789211B CN 103789211 B CN103789211 B CN 103789211B CN 201210432272 A CN201210432272 A CN 201210432272A CN 103789211 B CN103789211 B CN 103789211B
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Abstract
The present invention relates to a kind of abyssal sediment source Paecilomyces lilacinus A65 with anti-tealeaves pathomycete activity.This bacterium is Paecilomyces lilacinus, and preserving number is: CCTCC M 2012385, and itself has stronger restraining effect to tealeaves pathomycete, comprises and suppresses the sprouting of pathogenic bacterium spore and the growth of thalline.Bacterial strain provided by the invention can be mixed with wettable powder, for preventing and treating the morbidity of tealeaves common causative fungi.
Description
Technical field
The present invention relates to a strain and there is the pathogenic abyssal sediment source Paecilomyces lilacinus A65 of anti-tealeaves pathomycete.
Background technology
China is the native place of tealeaves, and Fujian is Main Tea Area, and Tea Pest is of a great variety, occurrence injury weight, and Pests of Tea-Plants reaches kind more than 390, disease for tea plant more than 100 is planted.Sick worm is caused harm and not only causes the tealeaves underproduction, quality decline, as tea place non-rational use of drug, pesticide residue exceed standard outstanding, pollute the environment, affect consumer health, also seriously hinder tea export, implementing the comprehensive regulation of disease worm is that development non-polluted tea is produced, and guarantees the important step of tealeaves high-quality, high yield and Sustainable development.At present, Chinese tea is Shi Chan tea big country in the world, but is not also produce tea power, has reason to be greatly exactly because chemical pesticide residue problem have impact on the outlet of tealeaves.For avoiding tea grower's blindly medication, should take in production to put prevention first, the strategy of integrated control, on the basis understanding disease and pest rule, taking many kinds of measures to prevent and treat, thus reaching economic, non-harmful prevention effect.Microbial pesticide has high-level efficiency, low residue to features such as person poultry harmless, is a kind of most suitable selection.
Paecilomyces lilacinus is common for preventing and treating various root knot nematode in existing research report, report from (1979) such as Jatata effective bacterial parasite that Paecilomyces lilacinus (Paecilomyces lilacinus) can be used as root-knot nematode egg and Cyst nematode ovum the earliest, in the test of laboratory, the embryo of Meloidogyne incognita ovum within 5 days, can be destroyed.Later many countries in succession test the prevention effect of Paecilomyces lilacinus but all only demonstrate Paecilomyces lilacinus has preventive and therapeutic effect to nematode, does not have further application development.Paecilomyces lilacinus can also produce abundant derivative, the similar indolylacetic acid product of the first, the growth of root system of plant and plant is promoted when its most significant physiological function is lower concentration, if therefore obviously nematode infection can not only be suppressed to chemical applied to plants, and the growth of plant nutrition organ can be promoted, also there is promoter action to the Germination and growth of seed simultaneously.
Thalassiomycetes is the important composition of marine microorganism, fungi by being adsorbed in, organic substance carries out the heterotrophic eukaryotic organisms of osmotrophy growth, with pre biooxidation ratio, fungi is relatively high, metabolic capacity is stronger, and vital movement is more complicated, and has very complicated ecological relationship with animals and plants in ocean again, it has the characteristic that a lot of Lu Sheng fungi does not have, and is the focus source of microbial preparation research and development in recent years.
Summary of the invention
Main purpose of the present invention is to provide a strain to have pathogenic abyssal sediment source Paecilomyces lilacinus (Paecilomyces lilacinus) A65 of anti-tealeaves pathomycete.It is by China typical culture collection center preservation (numbering: CCTCCM 2012385, preservation date on September 27th, 2012, place: Wuhan, China, Wuhan University)
Bacterial strain of the present invention is the Paecilomyces lilacinus in source, deep-sea, and fast, sporulation quantity is large, has stronger restraining effect to pathomycete common in tea growth in growth, can produce active prevention effect to tea place disease control.
Another object of the present invention is to provide the purposes of described Paecilomyces lilacinus A65, it is applied to and suppresses tealeaves pathomycete.Especially zonate spot source bacterium and anthrax pathogeny bacterium.
Another object of the present invention is the application method providing a kind of Paecilomyces lilacinus A65, its utilize described in the conidium of Paecilomyces lilacinus A65, add auxiliary agent and be mixed with wettable powder.
Another object of the present invention is to provide a kind of wettable powder suppressing tealeaves pathomycete, and its component comprises the conidium of aforesaid Paecilomyces lilacinus A65.
In preferred embodiment of the present invention, described wettable powder, comprises by weight:
Paecilomyces lilacinus A65 conidium 10%
Diatomite 70-80% and
Auxiliary agent 10-20%.
Wetting agent, dispersion agent, stablizer and the uv-protector etc. commonly used when auxiliary agent adopted in the present invention comprises this area preparation pulvis.
Present invention applicant is separated and obtains a fungal strain from Southwest Pacific abyssal sediment.Use ITS4, ITS5 primer amplification to obtain its ITS sequence, identify that this strain bacterium is Paecilomyces lilacinus (Paecilomyces lilacinus) through BLAST.Find in culturing process that it can produce conidium in a large number, find that its viable bacteria can suppress the growth of common tealeaves pathomycete and the sprouting of pathomycete spore preferably through screening active ingredients.The high cryptogam utilizing A65 fermentation to obtain adds the wettable powder that specific adjuvant can obtain having common tealeaves pathomycete biocontrol effect.
Accompanying drawing explanation
Fig. 1 is the colonial morphology of Paecilomyces lilacinus A65 on PDA flat board;
Fig. 2 is Paecilomyces lilacinus A65 form under the microscope;
Fig. 3 is the ITS electrophorogram of Paecilomyces lilacinus A65;
Fig. 4 is the inhibition of Paecilomyces lilacinus A65 to tealeaves pathomycete, wherein
A is zonate spot source bacterium inhibition, middle: tealeaves pathomycete; Both sides: A65;
B is anthrax pathogeny bacterium inhibition, middle: tealeaves pathomycete; Both sides: A65.
Embodiment
Embodiment 1
The isolation identification of A bacterial strain involved in the present invention
PDA+ paraxin+Streptomycin sulphate flat board is utilized to be separated 28 fungal strains from the abyssal sediment of seabed, 20 parts of Pacific Ocean, to be separated the tealeaves pathomycete from tealeaves scab, Phoma LH2 (Phoma sp.), tea zonate spot source bacterium LH13 (Pestalotiopsis theae), tea anthrax pathogeny bacterium LH30 (Colletotrichum gloeosporioides), Zuo Qiu chamber, Portugal bacterium LH107 (Neofusicoccum sp.), Alternaria LH129 (Alternaria sp.), it is indicator that plan dish crinosity belongs to LH162 (Pestalotiopsis sp.), dull and stereotyped face-off method is adopted to have detected the suppression tealeaves pathomycete activity of source, this 28 strain deep-sea fungi, wherein 1 strain filamentous fungus A65 shows good inhibition, it reaches 15.08mm to the suppression distance of LH13.
This strains A 65 is accredited as Paecilomyces lilacinus (Paecilomyceslilacinus) through means of taxonomic research and molecular biology research, and by China typical culture collection center preservation (numbering: CCTCC M 2012385, preservation date on September 27th, 2012, place: Wuhan, China, Wuhan University).
Adopt ordinary method, have studied the main cultural characters of this bacterium.This bacterium has the conidiophore of colyliform branch.Conidium majority slightly ovalize, the nearly fusiformis of minority or circle, unicellular, colourless.The optimum temperature range of growth and product spore is 25-30 DEG C; Suitable medium pH is 4-8.The substratum of potato, peptone, sucrose, nitrocalcite, Sodium phosphate dibasic composition is that the best and less expensive substratum of spore is produced in this bacteria growing and fermentation.
The microbial characteristic of this bacterium is: A65 fungi is very fast in the PDA slat chain conveyor speed of growth, and average every day can reach 8.36mm..Support 3 days, colony colour is light violet magenta, projection, surperficial opaque, and wheel line is obvious.Hyphae colorless, has barrier film.Conidiophore colyliform branch, 2-3 takes turns layer, and one deck tool 2-5 conidium stigma, stigma ampuliform or base portion slightly expand, and top is slightly elongated.Conidium chain raw, and the spore in a distant place is first ripe, and size is 2.75-3.00 × 1.6 μm, and monospore, colourless, majority are slightly in fusiformis, and minority is oval, and smooth surface, is shown in Fig. 1 and Fig. 2.
In PDA liquid culture, inoculate the ratio inoculation fermentation substratum of 1 piece of 6mm bacterium block in every 200ml, 28 DEG C, 180rpm shakes training 3 days visible white mycelium pellets and occur, the 4th day starts large volume production spore, produced spore by the 7th day and reach a little climax, produced spore at the 9th day subsequently and reach maximum.Along with the prolongation of incubation time, mycelium pellet finally becomes lilac from white, liquid nutrient medium color also correspondingly purpling grey.
The extracting genome DNA of B Paecilomyces lilacinus A65
(1) be inoculated on PDA flat board by the fungi of conservation or purifying, 28 DEG C of growths, after 5-7 days, are managed to EP with the aseptic a small amount of mycelium of toothpick picking, are put into liquid nitrogen and freeze 30-60min;
(2) take out freezing thalline, often pipe adds the CTAB buffer extract of 400 μ l, 65 DEG C of preheatings, is incubated 45-60min in 65 DEG C of shaking bath shaking tables;
(3) add isopyknic phenol: chloroform: primary isoamyl alcohol (25:24:1), fully mixes, the centrifugal 20-30min of 13000r/min, moves into new pipe mutually by upper water;
(4) (3) step is repeated;
(5) supernatant adds 0.7v Virahol, leaves standstill 30min or spend the night in 4 DEG C;
(6) the centrifugal 15min of 13000r/min, removing supernatant, precipitates by 70% washing with alcohol 2 times, dries up;
(9) with 20ul DDW dissolution precipitation ,-20 DEG C of preservations.
Get 5 μ L DNA and 1 μ L tetrabromophenol sulfonphthalein; Sepharose 0.8% detects its purity and concentration.
C identification of strains
By primer I TS45 '-TCC TCC GCT TAT TGA TAT GC-3 ' and ITS55 '-GGA AGT AAA AGTCGT AAC AAG G-3 ', A65 genomic dna is increased, PCR is 94 DEG C of denaturation 3min, 94 DEG C of sex change 1min, 53 DEG C of renaturation 45s, 72 DEG C extend 45s:32 circulation, 72 DEG C extend 10min, 4 DEG C of insulations.Amplified fragments T is carried Cloning Transformation to DH5 α.Run glue checking and obtain positive colony (see figure 3).
Positive colony checks order, and the ITS sequence obtaining Paecilomyces lilacinus A65 is as follows
TCCTCCGCTT ATTGATATGC TTAAGTTCAG CGGGTATTCC TACCTGATCC GAGGTCAACT 60
CTAAGAAAGT TGGGCGTTTT ACGGCGTGAC CGCCTCCGCG CTCCGGTGCG AGGTGTGTGC 120
TACTACGCAG GGGAGGCTGC GGCGGGGTCG CCACTGCATT TCGGGGGCGG CTGGTGTGCC 180
GTCCCCCAAC ACCGAGGCCC CGGGGGGGGC TCGAGGGTTG AAATGACGCT CGAACAGGCA 240
TGCCCGCCAG AATGCTGGCG GGCGCAATGT GCGTTCAAAG ATTCGATGAT TCACTGAATT 300
CTGCAATTCA CATTACTTAT CGCATTTCGC TGCGTTCTTC ATCGATGCCA GAACCAAGAG 360
ATCCGTTGTT GAAAGTTTTG ATTCATTTGT TTTTGCTTGT GCAACTCAGA GAAGAAATTC 420
CGCCCGCTGG GCGTAATGCA AGAGAGTTTG GGGTCCCTGC GGCGGGCGCC TGGGTCCGGC 480
GCCGGCGCGG GGGCAGGCGG CCGGGGCGTT CCCGCCGAGG CAACTGAGGT AAGGTTCACA 540
GTGGGTTTGG GAGTTGTATA ACTCGGTAAT GATCCCTCCG CTGGTTCACC AACGGAGACC 600
TTGTTACGAC TTTTACTTCC 620
Carry out BLAST analysis to the ITS sequence of Paecilomyces lilacinus A65, result shows this sequence and Paecilomyceslilacinus strain B59A(GENE Bank ID:HM242263.1) similarity is the highest, reaches 99%.Therefore can identify that this strain Pseudomonas is in Paecilomyces lilacinus (Paecilomyces lilacinus).
The restraining effect that embodiment 2 Paecilomyces lilacinus A65 is pathogenic to tealeaves pathomycete
Fungi Paecilomyces lilacinus A65 is inoculated into PDA substratum 28 DEG C and cultivates after 7 days, conidium is rinsed in dull and stereotyped with the aqua sterilisa being added with 0.1% tween-10, and load in EP pipe, hematimeter counts, and stepwise dilution spore concentration is to 1 × 10
6individual/ml is prepared into A65 spore suspension.Dull and stereotyped face-off method is adopted to measure A65 to the restraining effect of tealeaves pathomycete.
Dull and stereotyped face-off method: draw square crossing cross at flat plate bottom, places filter paper block in the dull and stereotyped distance center equidistant of PDA, paper block is inoculated the spore suspension of 20 μ l; At the instruction pathogenic bacteria bacterium cake of dull and stereotyped central authorities inoculation diameter 6mm, at 28 DEG C, cultivate 5-7d, to produce between Antagonistic Fungi and each indicator bacterium colony obvious antibacterial be with time measure antagonism bandwidth.Not inoculate Antagonistic Fungi, only connect indicator and contrast (Fig. 4).
The preparation of embodiment 3 Paecilomyces lilacinus A65 wettable powder
Single factor test assay method: utilizing unitary variant method to filter out suitable formulation carrier one by one from various carriers to be selected, auxiliary agent is diatomite, and other auxiliary agents are some.
Orthogonal experiment method: the method arranging multifactorial experiment by orthogonal table, is called orthogonal experimental design method.Its feature for: the experiment number 1. completed needed for test requirements document is few; 2. the distribution of data point is very even; 3. can analyze test-results with corresponding range analysis method, variance analysis method, regression analysis etc., draw valuable conclusion.
Orthogonal experiment design method orthogonal table arranges test.
Through experiment, finally show that optimum formula of the present invention be wettable powder agent prescription is 10% Paecilomyces lilacinus A65 conidium, 70%-80% diatomite, other auxiliary agents 10%-20%.
Claims (6)
1. abyssal sediment source Paecilomyces lilacinus (
paecilomyces lilacinus) A65, preserving number is: CCTCC M2012385.
2. the purposes of Paecilomyces lilacinus A65 as claimed in claim 1, is characterized in that, is applied to and suppresses tealeaves pathomycete.
3. the purposes of Paecilomyces lilacinus A65 as claimed in claim 2, it is characterized in that, described tealeaves pathomycete is zonate spot source bacterium or anthrax pathogeny bacterium.
4. an application method of Paecilomyces lilacinus A65, is characterized in that: the conidium utilizing the Paecilomyces lilacinus A65 described in claim 1, adds auxiliary agent and be mixed with wettable powder.
5. suppress a wettable powder for tealeaves pathomycete, it comprises the conidium of Paecilomyces lilacinus A65 according to claim 1.
6. a kind of wettable powder suppressing tealeaves pathomycete as claimed in claim 5, comprises by weight:
Paecilomyces lilacinus A65 conidium 10%
Diatomite 70-80% and
Auxiliary agent 10-20%.
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| CN102405939A (en) * | 2011-09-27 | 2012-04-11 | 德强生物股份有限公司 | Nematicidal combination comprising paecilomyces lilacinus and carbosulfan |
| CN102422844A (en) * | 2011-09-27 | 2012-04-25 | 德强生物股份有限公司 | Paecilomyces lilacinus-containing composition for preventing and controlling nematodes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102405939A (en) * | 2011-09-27 | 2012-04-11 | 德强生物股份有限公司 | Nematicidal combination comprising paecilomyces lilacinus and carbosulfan |
| CN102422844A (en) * | 2011-09-27 | 2012-04-25 | 德强生物股份有限公司 | Paecilomyces lilacinus-containing composition for preventing and controlling nematodes |
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| Mass production and commercial formulation of Paecilomyces lilacinus;Prabhu S 等;《Indian Journal of Nematology》;20081231;第33卷(第2期);131-133 * |
| 一株茶树内生淡紫拟青霉的鉴定及抗真菌活性研究;李绍锋 等;《生物灾害科学》;20120331;第35卷(第1期);第45页摘要，第45-46页第1节，第46-47页第2.1-2.3节 * |
| 淡紫拟青霉的特性与研究进展;张春龙 等;《湖北植保》;20120430(第2期);48-51 * |
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Effective date of registration: 20160115 Address after: 361000 No. 184, University Road, Xiamen, Fujian Patentee after: Oceanography Inst. No.3, State Bureau of Oceanography Patentee after: China Ocean Mineral Resources Research and Development Association Address before: 361000 No. 184, University Road, Xiamen, Fujian Patentee before: Oceanography Inst. No.3, State Bureau of Oceanography |