CN105638746A - Application of streptomyces lavendulae to preventing and treating dry rot of actinidia arguta - Google Patents

Application of streptomyces lavendulae to preventing and treating dry rot of actinidia arguta Download PDF

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Publication number
CN105638746A
CN105638746A CN201610053108.6A CN201610053108A CN105638746A CN 105638746 A CN105638746 A CN 105638746A CN 201610053108 A CN201610053108 A CN 201610053108A CN 105638746 A CN105638746 A CN 105638746A
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actinidia arguta
zucc
dry rot
arguta sieb
preventing
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李娟�
王旭
王东来
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Eastern Liaoning University
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Eastern Liaoning University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/10—Animals; Substances produced thereby or obtained therefrom

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  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Pest Control & Pesticides (AREA)
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  • Virology (AREA)
  • Agronomy & Crop Science (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

The invention discloses application of streptomyces lavendulae to preventing and treating dry rot of actinidia arguta and relates to the field of the agricultural biotechnology. The streptomyces lavendulae is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.9972. Sterile fermentation filtrate 100-fold diluent of the streptomyces lavendulae is directly sprayed to the stems of the actinidia arguta or poured to the roots of the actinidia arguta, and the prevention effect on dry rot of the actinidia arguta can reach 88.6%. The process is simple, cost is low, use is convenient, use of chemical pesticide in the cultivation process of the actinidia arguta can be reduced, the advantages of being safe and environmentally friendly are achieved, and good application and development prospects are achieved.

Description

The application in preventing and treating Actinidia arguta Sieb.et Zucc dry rot of the one strain Strepiomyces lavendulae
Technical field
The present invention relates to strain Strepiomyces lavendulae application in preventing and treating Actinidia arguta Sieb.et Zucc dry rot, belong to agricultural biological technical field.
Background technology
Actinidia arguta Sieb.et Zucc Actinidiaarguta (Sieb.etZucc.) Planch.ExMiq. has another name called Fructus Actinidiae Argutae, Radix actinidiae argutae, rattan melon, Fructus actinidiae chinensis, belongs to Actinidiaceae, the large-scale fallen leaves liana of Actinidia. Actinidia arguta Sieb.et Zucc fruit is less, smooth surface, and whole fruit is edible, nutritious, is a kind of emerging commodity fruit, is introduced and commerial growing by countries such as Canada, Chile, France, New Zealand and the U.S.. Domestic Actinidia arguta Sieb.et Zucc commercialization cultivation is just risen. At present, Dandong cultivated area has reached more than 10000 mu, is the maximum area of domestic Actinidia arguta Sieb.et Zucc cultivated area.
Over the past two years, Operation in Dandong Area Actinidia arguta Sieb.et Zucc plantation occurred in that a kind of new expression and Actinidia arguta Sieb.et Zucc dry rot. This disease occurs mainly in spring, and the butt portion of the Actinidia arguta Sieb.et Zucc that is injured forms auburn scab, and scab upwards expands, and cuts tissue of being injured open, and cortex deepens brown, and phloem separates with xylem, and there is white hypha on phloem surface. Identified, Actinidia arguta Sieb.et Zucc dry rot pathogen is Phomopsis fungus DiaporthenobilisSacc.&Speg.. This pathogen, mainly through Actinidia arguta Sieb.et Zucc basal part of stem cold injury site infection, causes that plant phloem and xylem are impaired, affects moisture, nutrient transport, causes plant strain growth gesture to die down and even wilts. This evil of being critically ill is serious, and Field diseases is about 20%, has a strong impact on Actinidia arguta Sieb.et Zucc yield.
This disease mainly adopts field management and Agro-chemicals control at present, but effect is undesirable. A large amount of use chemical pesticides make pathogen produce drug resistance, cause environmental pollution simultaneously. Utilize antagonistic microbe and tunning thereof that crop pest is carried out Biological control it can be avoided that secondary environmental pollution, it is believed that to be the green safety measure of control of plant disease.
Summary of the invention
It is an object of the invention to provide strain Strepiomyces lavendulae application in preventing and treating Actinidia arguta Sieb.et Zucc dry rot, to solve environmental pollution and the fruit Pesticide Residue that chemical pesticide control brings.
The object of the invention is achieved through the following technical solutions: strain Strepiomyces lavendulae application in preventing and treating Actinidia arguta Sieb.et Zucc dry rot, it is characterised in that the aseptic ferment filtrate of a strain Strepiomyces lavendulae is used for preventing and treating Actinidia arguta Sieb.et Zucc dry rot.
The preparation method of above-mentioned aseptic ferment filtrate is as follows: by Strepiomyces lavendulae inoculation in fluid medium, culture medium loading amount 70%, fermenter volume 30L, low whipping speed 150-200rpm/min, ventilation 15-20%, temperature 25-30 DEG C, under pH value 6.5 ~ 7.5 condition, carry out ferment at constant temperature cultivation, after fermenting 5 days, fermentation liquid 5000r/min centrifugal treating, preparing supernatant, supernatant adopts sterilised membrane filter to filter, and prepares aseptic ferment filtrate.
In order to check the above-mentioned aseptic ferment filtrate prevention effect to Actinidia arguta Sieb.et Zucc dry rot, carry out pot experiment. Result shows: 100 times of diluents of the aseptic ferment filtrate of Strepiomyces lavendulae adopt stem to spray all can reach good prevention effect with root irrigation, and preventive effect reaches 86.7%, 88.6% respectively.
Above-mentioned aseptic ferment filtrate is for preventing and treating the using method of Actinidia arguta Sieb.et Zucc dry rot: when using, and aseptic ferment filtrate after 1:100 and Shui Chong converts in proportion, is directly sprayed in Actinidia arguta Sieb.et Zucc stem or filled in Actinidia arguta Sieb.et Zucc root.
The one strain Strepiomyces lavendulae of the present invention carries out preservation at China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is: CGMCCNo.9972; Preservation address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, China Committee for Culture Collection of Microorganisms's common micro-organisms center, postcode 100101. Preservation date is: on November 14th, 2014.
Beneficial effects of the present invention: Actinidia arguta Sieb.et Zucc dry rot is had good prevention effect by the aseptic ferment filtrate of a strain Strepiomyces lavendulae provided by the invention, provides a kind of effective, nontoxic, noresidue, eco-friendly Biological control new way for the preventing and treating of Actinidia arguta Sieb.et Zucc dry rot.
Accompanying drawing explanation
Fig. 1 is the Strepiomyces lavendulae DTJ-24 phylogenetic tree based on 16SrDNA sequence construct of the present invention; Fig. 2 is the Strepiomyces lavendulae DTJ-24 of the present invention antagonism to Fructus Jujubae dried Chinese gooseberry maize ear rot pathogen.
Detailed description of the invention
Below in conjunction with preferred embodiment, technical scheme is set forth in. Following example are merely to illustrate and explain the present invention, and do not constitute the restriction to technical solution of the present invention.
The separation screening of embodiment 1 Antagonistic Fungi
1, purification is separated
In August ,-2015 in June, 2013, ground gathers pedotheque 50 parts to adopt five point samplings from Fengcheng City of Liaoning Province, Donggang City, wide pasture etc. The pedotheque gathered, natural air drying. Soil sampling 10g, is placed in the triangular flask equipped with 90ml sterilized water, shakes 20min on shaking table. After putting 30s, dilution 10 times successively, prepare 10-4Diluent. Micropipettor draws 10-4Diluent 0.1ml is to Gause I culture medium solid plate, and the coating of aseptic spreader is uniformly. 25 DEG C of constant temperature culture carton upside downs are cultivated 3-5 days. On picking flat board, the bacterial strain of different colonial morphologies is forwarded in Gause I culture medium and obtains pure culture, and sterilizing paraffin oil sealing is hidden, and 4 DEG C of preservations are standby. Gause I culture medium: soluble starch 20.0g, potassium nitrate 1.0g, sodium chloride 0.5g, K2HPO4��3H2O0.5g, MgSO4��7H2O0.5g, FeSO4��7H2O0.01g, agar 20.0g, water 1000ml, adjust pH value to 7.2.
2, screening
Primary dcreening operation: with Actinidia arguta Sieb.et Zucc dry rot pathogen Diaporthenobilis for indicator bacteria, adopts flat board face-off method to carry out Antagonistic Fungi primary dcreening operation. After indicator bacteria activation, prepare truffle with the aseptic card punch of diameter 0.6cm. Pathogen is inoculated in PDA plate central authorities, cultivates 24h for 25 DEG C. At the symmetrical inoculation in distance pathogen 3cm place test strains, not connect test strains for comparison, each process is repeated 3 times. It is placed in 25 DEG C of incubators and cultivates, cover with whole flat board to comparison, measure antibacterial bandwidth, select antibacterial obvious bacterial strain and carry out multiple sieve (Fig. 1). PDA culture medium: Rhizoma Solani tuber osi 200.0g, glucose 10.0g, agar 20.0g, water 1000ml, natural pH.
Multiple sieve: with Actinidia arguta Sieb.et Zucc dry rot pathogen Diaporthenobilis for indicator bacteria, adopt fermentation liquid growth rate method to carry out Antagonistic Fungi to sieve again: the Antagonistic Fungi that primary dcreening operation obtains is inoculated in Gause I fluid medium, 25 DEG C, rotating speed 160r/min, constant temperature oscillator is cultivated 3 days, fermentation liquid is centrifugal removes thalline, and after sterilised membrane filter filters, filtrate is standby. Take after filtrate 10ml and 90ml is cooled to the PDA mix homogeneously of 60 DEG C and be down flat plate, prepare pathogen truffle with diameter 0.6cm card punch, be inoculated in culture dish center, with sterilized water process for comparison. Each process is repeated 3 times, and 25 DEG C are cultured to comparison bacterium colony and cover with whole flat board, measurement processing colony diameter, obtain bacteriostasis rate. Filtering out the bacterial strain DTJ-24 that Actinidia arguta Sieb.et Zucc dry rot pathogen Diaporthenobilis has stronger antagonism, its bacteriostasis rate reaches 85.1%.
The qualification of embodiment 2 antagonistic strain
1, Morphological Identification and biochemical character
Colonial morphology: bacterial strain DTJ-24 aerial hyphae lilac, substrate mycelium in Gause I culture medium is colourless, can not lysochrome; Microscopic morphology: substrate mycelium is without barrier film under the microscope, does not rupture; Aerial hyphae multi-branched, and branch is shorter, spore filament length, spiral type, spore is oval to cylindricality, smooth surface.
Biochemical character: bacterial strain DTJ-24 can make gelatin liquefaction, and Starch Hydrolysis is weak. Generation hydrogen sulfide, energy decomposition of cellulose, milk peptonize, do not solidify. Glucose, fructose can be utilized, do not utilize sucrose, inositol, mannitol, Raffinose, D-xylose.
2,16SrDNA sequence analysis
Bacterial strain DTJ-24 is inoculated in Gause I fluid medium, and 25 DEG C of shaking tables cultivate 3d, centrifugal collection thalline. Sangon Biotech's bacterial genomes DNA extraction kit is adopted to extract the genomic DNA of this bacterial strain. Employing universal primer (27f:5 '-AGAGTTTGATCMTGGCTCAG-3 '; 1492r:5 '-TACGGYTACCTTGTTACGACTT-3 ') carry out the pcr amplification of 16SrDNA, amplification system (50ul): 1 �� TaqPCRMasterMix25 �� L, DNA profiling 1 �� L(0.1-10ng), primer (10 ��m of ol/L) each 2 �� L, ddH2O20 �� L. PCR reaction condition is: 94 DEG C of denaturation 4min; 94 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 2min, 30 circulations; 72 DEG C extend 10min, and 4 DEG C terminate reaction. Amplified production detects through 1% agarose gel electrophoresis, in-20 DEG C of Refrigerator stores, delivers to Sangon Biotech (Shanghai) Co., Ltd. and is purified, and two-way order-checking also splices output complete sequence.
The DNA sequence (see Seq1) that will obtain, is detected by the Blast in ncbi database and is downloaded the similar sequences and reference sequences arranged that check order. Use software ClustalX(Version1.83) multisequencing is compared. Choosing the 16SrDNA sequence of wherein 6 typical strains, with micromonospora kind Micromonosporaviridifaciens for outer group, the ortho position phase connection in operation analysis software Mega6.06 carries out phylogenetic tree structure. It is shown that bacterial strain DTJ-24 and Streptomyceslavendulae clusters in a branch (Fig. 2). Therefore, combining form, physiological and biochemical property and 16SrDNA Phylogenetic Analysis result, bacterial strain DTJ-24 is accredited as Strepiomyces lavendulae (S.lavendulae).
The preparation of the aseptic ferment filtrate of embodiment 3 Antagonistic Fungi
Bacterial strain DTJ-24 is inoculated in fluid medium, culture medium loading amount 70%, fermenter volume 30L, low whipping speed 150-200rpm/min, ventilation 15-20%, temperature 25-30 DEG C, under pH value 6.5 ~ 7.5 condition, carry out ferment at constant temperature cultivation, after fermenting 5 days, fermentation liquid 5000r/min centrifugal treating, preparing supernatant, supernatant adopts sterilised membrane filter to filter, and prepares aseptic ferment filtrate.
Liquid culture based formulas is as follows: yeast leaching powder 4.0g/L, Fructus Hordei Germinatus leaching powder 10.0g/L, glucose 4.0g/L, water 1000ml, pH value 7.5.
Embodiment 4 pot experiment
In order to check the bacterial strain DTJ-24 aseptic ferment filtrate biocontrol effect to Actinidia arguta Sieb.et Zucc dry rot, carry out this test. Testing and carry out in the 4-6 month in 2015, test Actinidia arguta Sieb.et Zucc Seedling life in 1 year, growth potential is homogeneous, and cultivation matrix is Actinidia arguta Sieb.et Zucc cultivation base soil, and culture apparatus is nutritive cube (specification 30cm �� 30cm).
1, Actinidia arguta Sieb.et Zucc is transplanted
Cut the earth from Actinidia arguta Sieb.et Zucc planting base, Dandong standby. Weigh appropriate soil, be mixed into 2% fertilizer, each nutritive cube loads 3.5 kilograms of mixed soil. Slightly compress after nutritive cube is filled substrate, then move into 1 strain Actinidia arguta Sieb.et Zucc Seedling, keep the skin wet as required, after 15d, treat that Actinidia arguta Sieb.et Zucc growth stably carries out follow-up test.
2, prepared by pathogen spore suspension
Actinidia arguta Sieb.et Zucc dry rot pathogen is inoculated in PDA test tube slant 27 DEG C cultivate 7d. Take cultured pathogen slant strains one, add 5ml sterilized water, gently the spore of agar surface is scraped, this spore suspension is placed in sterilizing 50ml triangular flask, several sterile glass balls placed in advance by bottle, is filtered with the absorbent cotton of sterilizing after shake well, and with aseptic water washing filtering residue 2 ~ 3 times, finally make filtrate volume reach 10.0ml, obtain spore suspension. Survey spore suspension concentration with blood counting chamber, finally prepare 5.0 �� 105The pathogen spore suspension of individual/ml.
3, the preparation of the aseptic ferment filtrate of Antagonistic Fungi: method is shown in embodiment 2.
4, pathogenic bacterial infection Actinidia arguta Sieb.et Zucc
Mark the long cross wound of 0.5cm by sterile razor blade at healthy test Actinidia arguta Sieb.et Zucc stem sample site, inoculate the pathogen spore liquid of preparation in 5ul step 2 with pipettor at Actinidia arguta Sieb.et Zucc trunk abraded area, cultivate 48h.
5, stem sprays preventive effect
Carry out stem with 100 times of diluents of aseptic ferment filtrate of step 3 preparation to spray, medicinal liquid is sprayed at uniformly stem, to drip for degree; Sterilized water sprays as negative control; 1000 times of diluents of Prochloraz spray as positive control. The strain of each process group 20, repeats for 3 times. Spraying medicine 1 time every 15 days, spray continuously 3 times, add up disease index, calculate prevention effect after 45d, result is in Table 1.
6, root irrigation preventive effect: carry out root irrigation with 100 times of diluent 100ml of aseptic ferment filtrate of step 3 preparation; Sterilized water processes as negative control; 1000 times of diluents of Prochloraz process as positive control. The strain of each process group 20, repeats for 3 times. Every medication in 15 days 1 time, processing 3 times continuously, add up disease index, calculate prevention effect after 45d, result is in Table 2.
The severity Scaling standard of Actinidia arguta Sieb.et Zucc dry rot trunk 0 grade: anosis; 1 grade: light brown speckle, scab average length��1.0cm occurs in morbidity portion; 3 grades: scab expands along trunk direction to both sides, and scab average length is at 1.1 ~ 5.0cm; 5 grades: typical scab occur, scab average length is in 5.1 ~ 9.0cm:7 level: scab expands, and average length is 9.1 ~ 15.0cm; 9 grades: wilting occurs in the blade of morbidity trunk, scab average length > 15.0cm.
Disease index=[�� (diseased plant numbers at different levels �� corresponding values at different levels)/(investigating the highest value of total strain number �� morbidity)] �� 100. Relative control effect (%)=[(comparison disease index processes disease index)/comparison disease index] �� 100.
Table 1 stem sprays prevention effect
Process Disease index Relative control effect (%)
Antagonistic Fungi DTJ-24 100 times of diluents of aseptic ferment filtrate 8.61��0.27 86.71��0.21a
1000 times of diluents of Prochloraz 7.57��0.44 88.31��0.54a
Sterilized water compares 64.81��0.23 �C
Note: in table, data are mean+SD, after data, same letter represents through Duncan duncan's new multiple range method inspection difference notable (P > 0.05)
Table 2 root irrigation prevention effect
Process Disease index Relative control effect (%)
Antagonistic Fungi DTJ-24 100 times of diluents of aseptic ferment filtrate 7.49��0.29 88.60��0.27a
1000 times of diluents of Prochloraz 6.92��0.37 89.47��0.35a
Sterilized water compares 65.74��0.17 �C
Note: in table, data are mean+SD, after data, same letter represents through Duncan duncan's new multiple range method inspection difference notable (P > 0.05)
From table 1,2,100 times of diluents of the aseptic ferment filtrate of bacterial strain DTJ-24 and 1000 times of diluents of chemical pesticide Prochloraz process compared with without obvious preventive effect difference, and prevention effect is good. Method of application two kinds different: stem sprays and all can reach good prevention effect with root irrigation. Therefore compared with using chemical pesticide, present invention process is simple, and cost is low, easy to use, it is possible to reduce the use of chemical pesticide in Actinidia arguta Sieb.et Zucc cultivation, has the advantage of safety and environmental protection and good application and DEVELOPMENT PROSPECT.

Claims (3)

1. strain Strepiomyces lavendulae application in preventing and treating Actinidia arguta Sieb.et Zucc dry rot.
2. the application described in claim 1, it is characterised in that the aseptic ferment filtrate of a strain Strepiomyces lavendulae is used for preventing and treating Actinidia arguta Sieb.et Zucc dry rot.
3. the aseptic ferment filtrate described in claim 2 is used for preventing and treating Actinidia arguta Sieb.et Zucc dry rot, it is characterised in that when using, and aseptic ferment filtrate after 1:100 and Shui Chong converts in proportion, is directly sprayed in Actinidia arguta Sieb.et Zucc stem or filled in Actinidia arguta Sieb.et Zucc root.
CN201610053108.6A 2016-01-26 2016-01-26 Application of streptomyces lavendulae to preventing and treating dry rot of actinidia arguta Pending CN105638746A (en)

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KR20100017997A (en) * 2010-01-08 2010-02-16 (주)씨엠씨코리아 A novel streptomyces lavendulae cmc0992[kctc18169p] active against plant fungal pathogens and preparation of microbial pesticide
CN103602607A (en) * 2013-07-31 2014-02-26 江西农业大学 One strain of streptomyceslavendulae X33 and applications thereof
CN103540556A (en) * 2013-11-12 2014-01-29 青岛明月蓝海生物科技有限公司 Streptomyces lavendulae and application of Streptomyces lavendulae to preparation of algae microbial fertilizer
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