CN107136122A - A kind of biocontrol agent for preventing and treating the late blight of potato - Google Patents

Abstract

The invention discloses a kind of biocontrol agent for preventing and treating the late blight of potato, belong to crop disease control technical field.The biocontrol agent by scab streptomycete (Streptomyceseuropaeiscabiei) St19 fermentation form, the bacterial strain on March 24th, 2014 China Committee for Culture Collection of Microorganisms's common micro-organisms center register preservation, preserving number is CGMCCNO.8962.The biocontrol agent can inhibition of potato late disease bacteria growth, preferable prevention effect is shown to the late blight of potato；It can use in potato seedling phase or Adult plant pouring root or be mixed and made into bacterial manure with organic fertilizer and use.The St19 bacterial strains are obtained from potato rhizosphere soil, compatible with soil ecology harmony, nontoxic, no pathogenicity, therefore with larger application potential in the biological control of the late blight of potato.

Description

A kind of biocontrol agent for preventing and treating the late blight of potato

Technical field

The present invention relates to a kind of biocontrol agent for preventing and treating the late blight of potato, belong to crop disease control technical field.

Background technology

By late disease bacteria（Phytophthora infestans (Mont)de Bary）Caused late blight is potato A kind of destructive disease in production, has generation in each potato main producing region in the world and popular, seriously threatens potato Production, its harm caused, difficulty of prevention and cure and exceedes rice blast and stripe rust of wheat to social influence, is considered as international First big crop disease.Late blight is universal and serious in China's generation, every year about 8,000,000,000 yuans caused by feeling late blight Economic loss, it has also become the potato production of limitation China and the first big obstacle for realizing industrialization.

So far, late blight preventing and treating is main or is used by cultivation disease-resistant variety and a large amount of of chemical pesticide.Wherein It is to prevent and treat the most successful measure of epidemic disease to cultivate disease-resistant variety, but the changeable different in nature feature of late disease bacteria toxicity composition tool, causes profit The disease-resistant variety resistance being bred as with main anti-gene is lost, and screens and cultivate in a short time and be with antilemic new varieties Extremely difficult.Although development of the chemical pesticide to agricultural played an important role, but the long-term use of chemical pesticide can be brought Many negative effects.Not only lack long-term validity using chemical pesticide, ecological environment destroyed, people and animals and beneficial also occur Biotoxication, the chemical substance residual quantity in grain and feed are significantly exceeded, the problems such as influence people and animals' life quality, are permitted Many countries and regions have prohibited some high poisons, the production of high residue chemical pesticide and used in succession.It is more next in current people The living environment of oneself is more paid close attention to, and is increasingly advocated the epoch of green living, exploring effective biological and ecological methods to prevent plant disease, pests, and erosion measure undoubtedly has weight Big theory and realistic meaning.

Actinomyces（Actinomycetes）Distribution is wide, and species is more, and metabolic function is different, is that a class has great value Microbial resources.Found at present from microorganism among about 8000 kinds of bioactive substances, nearly 70% is produced by actinomyces, Particularly streptomycete produce antibiotic be widely used in agricultural production, its have preventive effect time length, it is nontoxic, harmless, without residual Stay, it is pollution-free the advantages of, larger environmental benefit and economic benefit is had been achieved in agricultural production, with wide exploitation Prospect.Therefore, reach that efficient, environmentally friendly biological control purpose is ground as current agricultural science by screening and using Antagonistic Fungi One of focus studied carefully.

The content of the invention

It is an object of the invention to：Seriously occur for the current late blight of potato, and area expands year by year, chemical prevention Effect is undesirable, while there is provided a kind of biocontrol agent for preventing and treating the late blight of potato for the problem of bringing environmental pollution.

The purpose of the present invention can be achieved through the following technical solutions：

A kind of biocontrol agent for preventing and treating the late blight of potato, by scab streptomycete (Streptomyceseuropaeiscabiei) St19 fermentations are formed, and its preparation method is：

1）The preparation of seed liquor：Described scab streptomycete St19 is inoculated in Gause I fluid nutrient medium, 28 DEG C, 150 R/min, cultivates 5 d, seed liquor is made；

2）Fermentation：Seed liquor is inoculated in fermentation medium, fermentation condition is：1% ~ 2%vol of inoculum concentration, the mL of bottling amount 100 7.0 ~ 7.2,28 DEG C of/250 mL, fermentation medium initial pH value, 150 r/min, the d of fermentation time 9, obtains zymotic fluid；

3）The preparation of bacteria-free filtrate：The r/min of zymotic fluid 4000 is centrifuged after 5 min, with 0.45 μm of sterilizing filtering with microporous membrane Degerming, it is the biocontrol agent to obtain bacteria-free filtrate.

Wherein, step 2）The fermentative medium formula is：The wt of soluble starch 2%, the wt of glucose 1.5%, analysis for soybean powder 2% wt、MgSO₄·7H₂O 0.05% wt、K₂HPO₄ 0.1% wt、NaCl 0.1% wt、CaCO₃0.2% wt, remaining is distillation Water.

The scab streptomycete St19 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, protects The Tibetan date is on March 24th, 2014, and preserving number is CGMCCNO. 8962.

Its application mode has following two：

1）Used in potato seedling phase or Adult plant pouring root：In potato seedling phase or Adult plant continuously using 2 ~ 3 times, every time It is spaced 7 ~ 10d；

2）Bacterial manure is mixed and made into organic fertilizer to use.

Beneficial effect of the present invention：

The biocontrol agent of the present invention for preventing and treating the late blight of potato is capable of the growth of obvious inhibition of potato late disease bacteria.Due to It is biological agent, therefore without chemical pesticide using a series of problems brought, farmland pollution can be reduced, and can effectively prevent and treat horse Bell potato late blight.

Biocontrol actinomycetes used in the present invention are isolated from potato rhizosphere soil, compatible with soil ecology harmony, favorably In the advantage for giving full play to bacterial strain.

Biocontrol actinomycetes strain culturing condition used in the present invention is simple, it is easy to industrialized production, is opened with good Send out application prospect.

Brief description of the drawings

Fig. 1 is colonial morphologies of the bacterial strain St19 on Gause I culture medium；

Fig. 2 is bacterial strain St19 aerial hyphaes and spore filament shapes（400 under light microscope ×）；

Fig. 3 is bacterial strain St19 hypha forms（Electron microscope 5000 ×）；

Fig. 4 is the flat board fungistatic effect figure of bacterial strain St19 Antagonistic Fungi bacteria-free filtrate Phytophthora infestans.

Embodiment

For a better understanding of the present invention, the present invention is further elaborated with reference to the accompanying drawings and examples, but not It is limitation of the present invention.Experimental method used is conventional method unless otherwise specified in following embodiments.

The separation of the Antagonistic Actinomycetes of embodiment one

Detailed process is as follows：

1st, the collection of pedotheque

Experiment is taken back after the rhizosphere soil of different potato plot 5 ~ 10cm depths, packing mark are gathered from Fujian Province Zhouning County Room, after separation after natural air drying.

2nd, the separation of actinomyces

Separated using plate dilution method.The g of pedotheque 10 is weighed, three equipped with small bead and 90ml sterilized waters are poured into In the bottle of angle, 5 min are stood after 30 min of concussion, 10 times are diluted successively, 10 are configured to respectively^-2、10^-3、10^-4、10^-5、10^-6It is outstanding Supernatant liquid, each 0.1 ml of suspension for drawing various concentrations is added to Gause I culture medium（Add final concentration of 50 mg/L's K₂Cr₂O₇）The different single bacterium colony line purifying of picking after 28 DEG C of culture observations, 5 ~ 7 d is placed on flat board, after even spread.Will be pure Bacterial strain after change is transferred on Gause I slant medium and cultivated, and 4 DEG C save backup, and isolate and obtain 85 plants of actinomyces.

The bacterial strain St19 of embodiment two identification

1st, morphological feature is observed

By bacterial strain St19 streak inoculations on Gause I culture medium, after 28 DEG C of 10 d of culture, the form of bacterium colony is observed（Fig. 1）, Entered simultaneously with 45 DEG C of oblique cuttings after sterile cover slips, 28 DEG C of 7 d of culture, take out cover glass under light microscope and electron microscope Observe the morphological feature of mycelia（Fig. 2 and Fig. 3）.

2nd, cultural characteristic is observed

Using《Streptomycete identification handbook》The 8 kinds of culture mediums recommended, bacterium is observed after bacterial strain St19 is cultivated into 7 ~ 10 d in 28 DEG C Body growing state, records aerial hyphae, the color of substrate mycelium and whether produces soluble pigment.Bacterial strain St19 is in Gao Shi mono- Number culture medium, examine on Bake culture medium, starch ammonium culture medium, PDA culture medium, glucose yeast cream culture medium and oat medium Well-grown, the gentle raw hyphal development of substrate mycelium is good；And in number culture medium of kirschner and glucose asparagine culture medium Upper growth is poor, and yellow fade or brown colour fading soluble pigment (table 1) is produced on Gause I culture medium and PDA culture medium.Root Upper Morphometric analysis, compares streptomycete identification handbook, St19 is initially identified as into scab streptomycete according to this.

Cultural characteristics of the bacterial strain St19 of table 1 on different culture media

3rd, 16S rDNA sequence analyses

Genome extracts kit is extracted after bacterial strain St19 genomic DNAs, using universal primer F: 5'- AGAGTTTGATCCTGGCTCAG-3' and R:5'-GGTTACCTTGTTACGACTT-3', the PCR for carrying out 16S rDNA expands Increase.Reclaim pcr amplification product and obtain St19-16S rDNA, after connection, conversion, identification, positive colony send raw work biological work Journey（Shanghai）Nucleotide sequence information is sequenced to obtain in Co., Ltd, and sequence is 1548 bp（See sequence）.Gained sequence is carried It is sent to GenBank databases and carries out BLAST analyses and comparison, finds to belong to streptomycete with the higher bacterial strain of St19 homologys Category, wherein with bacterial strainStreptomyceseuropaeiscabieiSimilitude reaches 99%, and combining form feature and culture Feature, by bacterial strain St19 be accredited as scab streptomycete (Streptomyceseuropaeiscabiei)。

The optimization of the St19 strain fermentation process of embodiment three

Above-mentioned bacterial strains are inoculated in Gause I fluid nutrient medium, 28 DEG C, 150 r/min, after 5 d of culture, seed is made Liquid, is inoculated in fermentation medium.Using phytophthora infestans as target bacterium, pass through the screening and nutrition to carbon source, nitrogen source After condition single factor experiment and orthogonal test, the Optimal compositions of fermentation medium formula for obtaining the bacterial strain is：The wt of soluble starch 2%, The wt of glucose 1.5%, analysis for soybean powder 2% wt, MgSO₄·7H₂O 0.05% wt、K₂HPO₄ 0.1% wt、NaCl 0.1% wt、 CaCO₃0.2% wt, remaining is distilled water.Optimal conditions of fermentation is：The vol of inoculum concentration 1% ~ 2%, bottling amount 100mL/250mL, 7.0 ~ 7.2,28 DEG C of fermentation medium initial pH value, 150 r/min, the d of fermentation time 9.The r/min of zymotic fluid 4000 is centrifuged 5 It is degerming with 0.45 μm of sterilizing filtering with microporous membrane after min, obtain bacteria-free filtrate.

Example IV bacterial strain St19 bacteria-free filtrate bacteria inhibition assays

Relative bacteriostasis rate is determined using growth rate method, by bacteria-free filtrate with being cooled to 40 DEG C or so of rye culture medium by volume Than 1:10 mixings are down flat plate, to add the rye culture medium of sterilized water in proportion as control.Take that cell age is consistent, diameter 5mm late blights Germ silk block is placed in rye flat board center, is placed in 20 DEG C of insulating box and cultivates, and Timing measurement colony diameter after 15 d is calculated With respect to bacteriostasis rate, often processing is repeated 5 times, and as a result shows that bacterial strain St19 Phytophthora infestans has preferable antagonism （Fig. 4）.

The potted plant efficiency test of bacterial strain St19 zymotic fluids of embodiment five

Choose 3 ~ 5 leaf size late blights of potato height sense kind（Favorita）Seedling, 50 ml St19 bacterial strains hair is poured per young plant Zymotic fluid（About 10⁹CFU /ml）.Pouring root is inoculated with phytophthora infestans zoospore suspension after 24 h（5×10⁴Individual/mL）, often 20 ml of strain.It is inoculated with after 4 d and pours a 100 ml St19 bacterial strain fermentation liquors again.With 50% peace 1000 times of liquid of gram wettable powder Root irrigation is compareed as agricultural chemicals, and blank control is used as using the plant that clear water is handled.15 young plants of each processing, often handle 3 weights It is multiple.Investigation incidence daily after being ill pending, the statistics incidence of disease and disease index, until the clear water control incidence of disease reach 80% with Terminated experiment when upper.

Severity Scaling standard is as follows：

0 grade：Disease-free spot；

1 grade：Lesion area accounts for less than the 5% of whole blade area；

3 grades：Lesion area accounts for the 6%-10% of whole blade area；

5 grades：Lesion area accounts for the 11%-20% of whole blade area；

7 grades：Lesion area accounts for the 21%-50% of whole blade area；

9 grades：Lesion area accounts for more than the 50% of whole blade area.

Disease index（%）=[∑ disease grade blade number × represent numerical value)/(most grave illness series × total leaf number)] × 100%

Prevention effect（%）=[(blank control disease index-processing disease index)/blank control disease index] × 100%

Experimental result is shown in Table 2, and the result shows that for examination actinomyces St19 zymotic fluids the disease of the late blight of potato can be significantly reduced Feelings index, prevention effect is up to 74.65%, is just slightly below the prevention effect that chemical pesticide 50% pacifies 1000 times of liquid of gram wettable powder 80.22%.Greenhouse pot culture experimental result illustrates that St19 bacterial strains can effectively prevent and treat the late blight of potato, show good application Potentiality.

Prevention effect of the actinomyces St19 zymotic fluids of table 2 to the late blight of potato

Presently preferred embodiments of the present invention is the foregoing is only, all equivalent changes done according to scope of the present invention patent are with repairing Decorations, should all belong to the covering scope of the present invention.

SEQUENCE LISTING

<110>Inst. of Plant Protection, fujian Academy of Agricultural Science

<120>A kind of biocontrol agent for preventing and treating the late blight of potato

<130> 3

<160> 3

<170> PatentIn version 3.3

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<211> 20

<212> DNA

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agagtttgat cctggctcag 20

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<212> DNA

<213> R

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ggttaccttg ttacgactt 19

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<211> 1548

<212> DNA

<213> St19-16S rDNA

<400> 3

agcttgcatg cctgcaggtc gacgattaga gtttgatcct ggctcaggac gaacgctggc 60

ggcgtgctta acacatgcaa gtcgaacgat gaaccacttc ggtggggatt agtggcgaac 120

gggtgagtaa cacgtgggca atctgccctt cactctggga caagccctgg aaacggggtc 180

taataccgga tacaacactc tcgggcatcc gatgagtgtg gaaagctccg gcggtgaagg 240

atgagcccgc ggcctatcag cttgttggtg aggtaacggc tcaccaaggc gacgacgggt 300

agccggcctg agagggcgac cggccacact gggactgaga cacggcccag actcctacgg 360

gaggcagcag tggggaatat tgcacaatgg gcgaaagcct gatgcagcga cgccgcgtga 420

gggatgacgg ccttcgggtt gtaaacctct ttcagcaggg aagaagcgaa agtgacggta 480

cctgcagaag aagcgccggc taactacgtg ccagcagccg cggtaatacg tagggcgcga 540

gcgttgtccg gaattattgg gcgtaaagag ctcgtaggcg gtctgtcgcg tcgaatgtga 600

aagcccgggg cttaaccccg ggtctgcatt cgatacgggc agactagagt gtggtagggg 660

agatcggaat tcctggtgta gcggtgaaat gcgcagatat caggaggaac accggtggcg 720

aaggcggatc tctgggccat tactgacgct gaggagcgaa agcgtgggga gcgaacagga 780

ttagataccc tggtagtcca cgccgtaaac ggtgggaact aggtgttggc gacattccac 840

gtcgtcggtg ccgcagctaa cgcattaagt tccccgcctg gggagtacgg ccgcaaggct 900

aaaactcaaa ggaattgacg ggggcccgca caagcagcgg agcatgtggc ttaattcgac 960

gcaacgcgaa gaaccttacc aaggcttgac atacaccgga aacggccaga gatggtcgcc 1020

cccttgtggt cggtgtacag gtggtgcatg gctgtcgtca gctcgtgtcg tgagatgttg 1080

ggttaagtcc cgcaacgagc gcaacccttg ttctgtgttg ccagcatgcc cttcggggtg 1140

atggggactc acaggagact gccggggtca actcggagga aggtggggac gacgtcaagt 1200

catcatgccc cttatgtctt gggctgcaca cgtgctacaa tggcaggtac aatgagctgc 1260

gaagccgtga ggcggagcga atctcaaaaa gcctgtctca gttcggattg gggtctgcaa 1320

ctcgacccca tgaagtcgga gttgctagta atcgcagatc agcagtgctg cggtgaatac 1380

gttcccgggc cttgtacaca ccgcccgtca cgtcacgaaa gtcggtaaca cccgaagccg 1440

gtggcccaac cccttgtggg agggagctgt cgaaggtggg actggcgatt gggacgaagt 1500

cgtaacaagg taccaatctc tagaggatcc ccgggtaccg agctcgaa 1548

Claims (4)

1. a kind of biocontrol agent for preventing and treating the late blight of potato, it is characterised in that described biocontrol agent is by scab streptomycete (Streptomyceseuropaeiscabiei) St19 fermentation form, its preparation method is：

1）The preparation of seed liquor：Described scab streptomycete St19 is inoculated in Gause I fluid nutrient medium, 28 DEG C, 150 R/min, cultivates 5 d, seed liquor is made；

2）Fermentation：Seed liquor is inoculated in fermentation medium, fermentation condition is：1% ~ 2%vol of inoculum concentration, the mL of bottling amount 100 7.0 ~ 7.2,28 DEG C of/250 mL, fermentation medium initial pH value, 150 r/min, the d of fermentation time 9, obtains zymotic fluid；

3）The preparation of bacteria-free filtrate：The r/min of zymotic fluid 4000 is centrifuged after 5 min, with 0.45 μm of sterilizing filtering with microporous membrane Degerming, it is the biocontrol agent to obtain bacteria-free filtrate.

2. a kind of biocontrol agent for preventing and treating the late blight of potato according to claim 1, it is characterised in that step 2）It is described Fermentative medium formula is：The wt of soluble starch 2%, the wt of glucose 1.5%, analysis for soybean powder 2% wt, MgSO₄·7H₂O 0.05% wt、K₂HPO₄ 0.1% wt、NaCl 0.1% wt、CaCO₃0.2% wt, remaining is distilled water.

3. a kind of biocontrol agent for preventing and treating the late blight of potato according to claim 1, it is characterised in that the scab chain Mould St19 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preservation date is in March, 2014 24, preserving number was CGMCCNO. 8962.

4. a kind of biocontrol agent for preventing and treating the late blight of potato according to claim 1, it is characterised in that its application mode Have following two：

1）Used in potato seedling phase or Adult plant pouring root：In potato seedling phase or Adult plant continuously using 2 ~ 3 times, every time It is spaced 7 ~ 10d；

2）Bacterial manure is mixed and made into organic fertilizer to use.