CN107779419B

CN107779419B - Streptomyces cinnamoneumochromogenes ZX6 for preventing and treating sunflower sclerotiniose and application thereof

Info

Publication number: CN107779419B
Application number: CN201710330364.XA
Authority: CN
Inventors: 张建丽; 郑晓薇; 单双权; 郭靖楠; 庄俊丽
Original assignee: Beijing Institute of Technology BIT
Current assignee: Beijing Institute of Technology BIT
Priority date: 2017-05-11
Filing date: 2017-05-11
Publication date: 2020-09-04
Anticipated expiration: 2037-05-11
Also published as: CN107779419A

Abstract

The invention discloses a streptomyces cinnamoneumochromogenes ZX6 for preventing and treating sunflower seedling-stage sclerotinia, the preservation number of the strain is CGMCC 13116, and the strain belongs to the technical field of microorganisms. Experiments such as separation activation and identification of pathogenic sclerotinia sclerotiorum of sunflower sclerotinia sclerotiorum, primary screening and secondary screening experiments of antagonistic bacteria, identification and analysis of antagonistic bacteria strains and the like show that the strain ZX6 has strong capability of inhibiting sclerotinia sclerotiorum hypha growth. The incidence of infection of sunflower seed hulls treated by the strain ZX6 zymogen liquid is reduced to 40%, the promotion rate of the strain ZX6 fermentation filtrate on seed germination is 51.1%, the prevention and treatment effect of in vitro leaves of the strain ZX6 zymogen liquid is 54%, the prevention and treatment effect of the strain ZX6 zymogen liquid potting experiment is 58.3%, and the growth and the propagation of sclerotinia sclerotiorum can be effectively inhibited. The strain has simple culture conditions, easy preservation, environmental protection and good development and application values.

Description

Streptomyces cinnamoneumochromogenes ZX6 for preventing and treating sunflower sclerotiniose and application thereof

Technical Field

The invention relates to screening identification and application of streptomyces cinnamoneumoniensis (Streptomyces cinnamunensis) ZX6 for preventing and treating sunflower sclerotiniose, in particular to primary application of the strain in prevention and treatment of sunflower sclerotiniose, and belongs to the technical field of microorganisms.

Background

The sunflower sclerotiniose is one of the most important diseases of the sunflower, is generated in all countries in the world, has strong pathogenicity of pathogenic Sclerotinia sclerotiorum (sclerotiotium sclerotiorum), can penetrate through the epidermis of a host plant to kill cells, further infects a plurality of parts of roots, stems, leaves, flower discs and the like of the sunflower, even causes the whole or partial rot and death of the plant, and can cause great loss to the sunflower if the management is not proper. In recent years, the steady development of sunflower planting has been greatly affected by the widespread occurrence and serious harm of sunflower sclerotinia.

Sunflower sclerotinia rot is also known as white rot or rotten disc disease. Sunflower sclerotiniose can be divided into a mycelium infection type and an ascospore infection type from the infection form of pathogenic bacteria; according to the occurrence part and symptoms of sclerotinia, the sclerotinia rot type fungi can be divided into root rot type fungi, stem rot type fungi, leaf rot type fungi and disc rot type fungi, wherein the root rot type fungi is caused by infecting sclerotium in soil or seeds in the form of mycelium after germination; the primary infection sources of the stem rot type, the leaf blight type and the rotten disc type are ascospores, and the infection capacities of the mycelia and the ascospores are obviously different under different ecological conditions.

The pathogenic bacteria of sunflower sclerotinia sclerotiorum, namely sclerotinia sclerotiorum, is soil inhabitation bacteria, can survive and accumulate in soil for a long time, and 90 percent of the life history of the sclerotinia sclerotiorum can survive in the soil for a long time in the form of sclerotia. The sclerotium can be dormant on the surface layer of the soil or mixed in seeds, has strong vitality, can survive for 6 to 8 years in dry soil and can survive for 3 to 5 years in humid soil. The optimal temperature range of sclerotinia sclerotiorum hypha growth is 20-28 ℃, the optimal pH value is 4-7, the optimal carbon source and nitrogen source are mannose and asparagine respectively, the hypha growth is most sensitive to Cu and Fe deficiency, and the hypha can grow vigorously and uniformly on natural and semi-natural culture media such as PDA, PSA and the like. The hyphae are treated at 45 deg.C for 10min, and the sclerotium is treated at 50 deg.C for 10min, without survival. Most of the serious disease areas of sunflower sclerotium diseases in China are rainy in the late growth period (7-8 months), so that pathogenic bacteria are mainly infected by ascospores. The disease resistance of the overground parts of the sunflowers is different, the leaves are stronger than the stems, the stems are stronger than the flower discs, the rotten disc type sclerotinia is the most serious, the root rot type and the stem rot type are the second, the leaf withering type rarely occurs, the sunflower seeds only occur on partial leaves, certain scattered light is needed for the formation of the ascomycetes discs, and the ultraviolet irradiation can stimulate the germination of sclerotia. After dark and natural light treatment, the sclerotium only generates an ascon disc handle, but does not form an ascon disc, and the ascon disc has obvious phototropism. If no exogenous nutrition exists, the sclerotium can germinate to produce the ascochyta as long as certain humidity and illumination are ensured. The sclerotium producing ascomycete plate can be divided into a needle-shaped period, an expansion period and a funnel period which are 3 periods, the proper temperature for sclerotium to germinate to form the ascomycete plate is 18-20 ℃, the high temperature is not easy to germinate, the germination of sclerotium needs proper ventilation conditions, and the number of ascomycete plates growing by covering soil of 0-1cm is the largest generally.

In the aspect of prevention and treatment, because the disease-resistant breeding progress of the sunflower sclerotinia sclerotiorum is slow, no disease-resistant variety and high-efficiency disease-resistant variety exist at present. The chemical prevention and control mainly uses carbendazim, prochloraz, dimethachlon and other medicaments, but the sunflower plants are too tall and large, the field medication is very difficult, and the effect of the medicaments on the sclerotium in the soil is limited, so the prevention and control effect is not ideal. In the aspect of agricultural control, an economical, practical and effective method is also lacking. Therefore, screening and utilizing beneficial microorganisms and metabolites thereof to prevent and treat sunflower sclerotinia sclerotiorum is a specific measure for implementing green, environmental protection and safety.

Disclosure of Invention

The invention aims to provide a disease-preventing growth-promoting strain capable of well controlling the occurrence of sunflower sclerotinia sclerotiorum diseases and application thereof.

The invention collects soil samples from inner Mongolia, Qinghai and spring regions, obtains single colonies of different strains on a culture medium of Gao 'S I, improved Gao' S II, TSA, LB, ISP2 and PDA by a dilution coating plate method, preliminarily screens out the strain with obvious inhibition effect on the sclerotinia sclerotiorum of sunflower by adopting a plate antagonism experiment, determines the species to which the strain belongs by 16S rRNA gene sequence, has no pathogenic carcinogenicity on human and livestock, and can be safely used for biological prevention and control of the sclerotinia sclerotiorum of sunflower.

The technical scheme provided by the invention is that the Streptomyces cinnamoneumoniae ZX6 for preventing and treating sunflower sclerotiniose and the application thereof, according to morphological characteristic observation and 16S rRNA gene sequence analysis, the strain is determined to be Streptomyces cinnamoneumoniae (Streptomyces cinnamonensis), and is named as Streptomyces cinnamoneumoniae ZX6(Streptomyces cinnamonensis ZX 6). The strain is preserved in the China general microbiological culture Collection center in 2017, 4 and 10 months, with the preservation number of CGMCC 13116 and the preservation unit address of: xilu No. 1 Hospital No. 3, North City, rising to the sun.

The strain ZX6 can grow on different culture media such as Gao's number one, ISP2, LB and PDA. The strain ZX6 grows well on the Gao's first culture medium, the colony is small, the hypha in the medium is beige, the aerial hypha is pink white at the early stage and becomes light gray at the later stage, and no soluble pigment is generated; the strain is inoculated on an ISP2 culture medium, the growth condition is good, the middle is raised, the colony is large, the hypha in the medium is white, the aerial hypha is pink, and light pink pigment is generated; the bacterial colony of the strain on an LB culture medium is small, has wrinkles and is wet, the hypha in the medium is light gray, the aerial hypha is brown, and no soluble pigment is generated; the colony on the PDA culture medium has folds, is large, the hypha in the medium is white, the aerial hypha is brown chocolate color, and no soluble pigment is generated. The growth was good at 28-30 ℃, the strain ZX6 was gram positive when observed under a microscope by gram staining, and the shape of the spores was oblong.

The screening and identifying method is applied by utilizing fermentation liquor and fermentation liquor extract of the strain ZX 6.

The invention provides a screening and identifying method of various antagonistic bacteria to sclerotinia sclerotiorum, and mainly relates to an antagonistic experiment, an in-vitro leaf control experiment, a pot culture experiment and the like of fermentation liquor of a strain ZX6 on sclerotinia sclerotiorum.

The invention has the following beneficial effects:

the strain ZX6 related by the invention is identified as streptomyces cinnamoneumoniensis (Streptomyces cinnnamonensis), has no pathogenic carcinogenicity to human and livestock, and is antagonistic actinomycetes capable of safely preventing and treating sunflower sclerotiniose. The application example shows that the pot experiment shows that the prevention and treatment effect of the strain on the sclerotinia rot of the sunflower seedling stage reaches 58.3%. At present, chemical prevention and control methods are mainly adopted for preventing and controlling sunflower sclerotiniose at home and abroad, mainly used chemical prevention and control methods comprise carbendazim, prochloraz, dimethachlon and the like, but sunflower plants are too tall and difficult to apply in fields, and the effect of the chemical on sclerotinia in soil is limited, so the prevention and control effect is not ideal. The inhibition effect of streptomyces cinnamoneumoniae on sunflower sclerotinia sclerotiorum has not been reported at home and abroad. Therefore, the strain ZX6 has good development potential and application value for preventing and treating sunflower sclerotinia rot.

Drawings

FIG. 1 is a graph showing the results of plate confrontation experiments of Streptomyces cinnamoneumoniae ZX6(Streptomyces cinnnanensis ZX6) in the primary screening of the strain ZX6 in example II.

FIG. 2 is a graph showing the growth results of strain ZX6 in the identification of strain ZX6 in example two.

FIG. 3: the result chart of ZX6 phylogenetic tree construction in 16S rRNA gene sequence identification and sequence analysis in example II.

FIG. 4 is a graph showing the effect of ZX6 bacterial liquid on seed germination in the sunflower seed germination experiment in example III (original strain ZX6 original bacterial liquid, original 20 strain ZX6 original bacterial liquid diluted 20 times, filtrate of strain ZX6 fermentation liquid, and filtrate of 20 diluted 20 times).

FIG. 5 is a result chart of an in vitro leaf control re-screening experimental group of the strain ZX6 in the third embodiment.

FIG. 6 is a graph showing the results of the control group of the in vitro leaf-leaf double-screening control of the strain ZX6 in the third example.

FIG. 7 is a graph showing the results of the original bacterial liquid experimental group in the sunflower potting experiment in the third example (the left is a full root infection graph, and the right is a partial root infection graph).

FIG. 8 is a graph showing the results of the blank control group in the sunflower potting experiment in the third example.

FIG. 9 is a graph showing the results of the control group in the sunflower potting experiment in the third embodiment.

Detailed Description

The following examples are provided to further illustrate the invention.

EXAMPLE screening and identification of Sclerotinia sclerotiorum

The sclerotinia sclerotiorum strain of the invention is separated from sunflower straws in inner Mongolia areas. Cutting sunflower straws picked from an inner Mongolia area with a knife, taking Sclerotinia sclerotiorum out of the sunflower straws, sterilizing the sunflower straws with 75% ethanol, washing the sunflower straws with sterile water for 2-3 times, placing the sunflower straws in a potato glucose agar culture medium, culturing the sunflower straws at the constant temperature of 25 ℃ for 3-5 days until hyphae grow out, picking the hyphae until a new potato glucose agar culture medium grows for standby, and carrying out ITS zone sequence determination on the purified Sclerotinia sclerotiorum to determine the Sclerotinia sclerotiorum (Lib.) by de Bary).

EXAMPLE two preliminary screening and identification of Strain ZX6

The method for screening the strains for preventing and treating the sunflower sclerotinia sclerotiorum comprises the following steps:

first, collection of soil sample

5 parts of soil sample were randomly collected from each of inner Mongolia, Qinghai and Jiuquan regions. Removing surface soil, collecting soil sample of about 300g at depth of 5-20cm, subpackaging, labeling, and returning to laboratory.

Secondly, purifying and culturing the isolated strain

Separating the strain by dilution coating plate method, weighing 1g of soil sample, mixing in 9mL of sterile water as 10^-1Diluting the solution in a gradient manner; are respectively used 10^-2、10^-3And 10^-4Sample dilutions were plated on individual isolation medium plates, 2 plates were plated per dilution, and each plate was plated at 0.2 mL. The surface of the agar plate must be dry in order for the inoculum to be absorbed immediately; the separation plate is inversely cultured for 1-4 weeks at 28 ℃, and periodically checked every 3-4 d; by morphological comparison, distinguishing strain with obviously inconsistent morphology, inoculating the strain in Gao's No. one culture medium (soluble starch 20g, NaCl 0.5g, KNO)₃1g,MgSO₄.7H₂O 0.5g, K₂HPO₄0.5g,FeSO₄.7H₂0.01g of O, 20g of agar, 1000ml of distilled water, pH7.2), modified No. II Goodpasture medium (1 g of glucose, Na₂HPO₄0.01g, tryptone 0.3g, vitamin complex 0.2ml, agar 20g, distilled water 1000ml, pH7.2), LB medium (tryptone 10g, yeast extract 5g, agar 20g, distilled water 1000ml, pH7.2), tryptone soy agar medium (TSA) (tryptone 15g, soy peptone 5g, sodium chloride 5g, agar 20g, distilled water 1000ml, pH7.2), glucose yeast medium (Bacto-yeast extract 4g, Bacto-malt extract10g, glucose 4g, agar 20g, distilled water 1000ml, pH7.2) and potato glucose agar medium (PDA) (potato extract 3g, glucose 20g, yeast extract10g, agar 20g, distilled water 1000ml, pH7.2) were grown up for 3-5d at 28 ℃ in an inverted culture.

Thirdly, primary screening of antagonistic strains

Screening effective antagonistic strains by adopting a plate opposing method, beating a bacterium block with the diameter of 6mm around an activated sunflower sclerotinia sclerotiorum colony, inoculating the bacterium block with the diameter of 6mm around a potato glucose agar culture medium, inoculating antagonistic bacterium cakes with the diameter of 3-5d cultured around the sclerotinia sclerotiorum block at equal intervals, culturing in a constant-temperature incubator at 25 ℃ for 3-5d, observing, measuring and recording the diameter of a bacteriostatic ring, and repeating the steps for 3 times.

Fourth, identification of Strain ZX6

1. Morphological characteristics of culture

Strain ZX6 grew normally on Gao's I medium, glucose yeast medium, LB and potato dextrose agar medium. The strain ZX6 grows well on the Gao's first culture medium, the hypha in the medium is beige, the aerial hypha is pink white at the early stage, and becomes light gray at the later stage; inoculating the strain ZX6 on a glucose yeast culture medium, wherein the growth condition is good, the middle bulge is formed, the hypha in the medium is white, and the aerial hypha is pink; the bacterial colony of the strain on an LB culture medium is wrinkled and moist, the hypha in the medium is light gray, and the aerial hypha is brown; the bacterial colony on the potato dextrose agar culture medium has folds, the hypha in the culture medium is white, and the aerial hypha is brown chocolate; the growth was good at 28-30 ℃, and the strain ZX6 was gram-positive and the spore shape was oblong when observed under a microscope by gram staining. No soluble pigment was produced on any of the other 3 media except that there was light pink pigment production on the glucose yeast medium. The morphological results are shown in FIG. 1.

2. Physiological and biochemical experiment

Specifically, according to the manual of identifying common bacteria systems and the manual of identifying streptomycete, the identification is carried out through physiological and biochemical experiments: the strain ZX6 is gram-positive bacteria, and the results of physiological and biochemical experiments are shown in the following table.

TABLE 1 physiological and biochemical Properties of Strain ZX6

Note: "+": the reaction is positive or can be grown and utilized; "-": the reaction is negative or can not grow and be utilized.

3. 16S rRNA gene sequence identification

16S rRNA gene sequence identification is carried out on a strain ZX6 (shown in figure 2) with better antagonistic effect to determine the species to which the strain belongs, and the strain is identified to be Streptomyces cinnamoneumoniensis (Streptomyces cinnamonensis).

3.1 extraction of DNA

Total DNA was extracted in small quantities mainly by the methods of Kim et al (1995) and Rainey et al (1996).

(1) Picking thallus from the plate or the inclined plane, centrifuging for 3min at 12000r/min with sterile water, and washing for 2 times; the resulting mixture was further centrifuged and washed 1 time with TE Buffer under the same conditions.

(2) mu.L of TE (pH 8.0) buffer was added to suspend the cell pellet, 30. mu.L of 20% SDS and 3. mu.L of 20mg/mL proteinase K were added thereto and mixed gently, followed by water bath at 30 ℃ for 1 hour.

(3) Add 100. mu.L of 5M NaCl and mix well, add 80. mu.L of CTAB/NaCl (10%/0.7M) solution and mix gently, water bath at 65 ℃ for 20 min.

(4) Adding 800 μ L phenol/chloroform/isoamyl alcohol (25:24:1), mixing, centrifuging at 12000r/min for 5min, transferring the supernatant to a new centrifuge tube, and extracting twice.

(5) Transferring the supernatant to a new centrifuge tube, adding isopropanol with the volume of 0.6 time, gently mixing, and centrifuging at 12000r/min for 15 min.

(6) The supernatant was discarded, 1mL of 70% ethanol was added, the mixture was gently mixed, and the mixture was centrifuged at 12000r/min for 15 min.

(7) The supernatant was discarded, air-dried, 50. mu.L of sterile water was added to dissolve the DNA, and the mixture was stored at 4 ℃.

3.216S rRNA Gene sequence identification

PCR amplification was performed using the 16S rRNA gene universal primer, 27f for the forward primer PA (corresponding to bases 8-27 of the E. coli 16SrRNA gene) 5'-AGAGTTTGATCCTGGCTCAG-3', 1525r for the reverse primer PB (corresponding to bases 1492-1514 of the E. coli 16SrRNA gene) 5'-AGAAAGGAGGTGTACCAGCC-3', 50. mu.L for the forward and reverse primers, 1.0. mu.L each, 2. mu.L for the DNA template, 2 × Mastermix 25. mu.L, ddH₂O was added to 50. mu.L. PCR amplification conditions: pre-denaturation at 95 ℃ for 5min, annealing at 54 ℃ for 1min, extension at 72 ℃ for 1.5min, 35 cycles in total, extension repair at 72 ℃ for 10min, and reaction termination at 4 ∞. The PCR product was purified by Wizard PCR Purification System (Promega) according to the procedure recommended in the specification. PCR products were determined by Beijing Nosai Gene IncThe sequence is 1427bp (shown as a base sequence of a ZX 616S rRNA gene). Comparing the sequence of the 16S rRNA gene with the sequence in GenBank/EMBL/DDBJ database, finding out the representative sequence of similar bacteria published in related species from the database, after multiple alignment of the obtained sequences, adopting CLUSTAL X2.0 software and MEGA version5 software to construct a phylogenetic tree of a strain ZX6 by using an ortho-junction method (figure 3), and finding that the gene has very high consistency (100%) with the 16S rRNA gene of Streptomyces cinnamonensis (Streptomyces cinnnnanensis) according to morphological characteristics, physiological and biochemical experiments and the like, thereby indicating that the strain ZX6 is the Streptomyces cinnamonensis.

Example rescreening and Primary application of three Strain ZX6

Sunflower seed husk experiment

The strain ZX6 was inoculated into a seed medium (2 g of soluble starch, 0.3g of casein enzymatic hydrolysate, 0.5g of glucose, 0.2g of Bacto-yeast extract, 0.5g of Bacto-malt extract, 0.3g of calcium carbonate, 100ml of sterile water, pH7.2) sterilized at 121 ℃ under 0.1MPa, and cultured in a shaking incubator at 170rpm/min at 29 ℃ for 3-5 days. According to the inoculation amount of 5% (namely 10ml of seed culture medium is added into 190ml of corresponding culture medium), the strain ZX6 in the seed culture medium is inoculated into a Gao's No. I liquid culture medium (namely agar is not added into a Gao's No. I solid culture medium), and then the strain is put into a full-temperature shaking incubator for culture, the rotation speed is kept at 170rpm/min, the temperature is controlled at 29 ℃, and the strain is cultured for 3-5 d.

Treating a strain ZX6 zymocyte liquid: taking out the strain ZX6 zymocyte liquid put in a constant-temperature shaking incubator, adding the strain ZX6 zymocyte liquid into a 50ml centrifuge tube, carrying out high-speed centrifugation at the rotating speed of 12000r/min for 20min, sucking supernatant liquid by using a disposable sterile syringe, filtering by using a hydrophilic polyether sulfone membrane to obtain filtrate without strain ZX6 hyphae, finally diluting the strain ZX6 original strain liquid and the filtrate by 20 times respectively, and finally obtaining the original strain liquid, the original strain liquid diluted by 20 times, the filtrate and the filtrate diluted by 20 times.

Primarily treating sunflower seeds: soaking sunflower seeds in sterile water for 2min, removing sterile water, adding 75% ethanol solution, soaking for 1min, washing once, removing ethanol solution, adding 2% sodium hypochlorite solution, soaking for 2min, soaking twice, washing in sterile water for 1 min.

And (3) retreatment of sunflower seeds: dividing the treated seeds into five parts, adding a proper amount of sterile water into one part, adding an equal amount of original bacteria liquid into the other part, adding an equal amount of diluted 20-fold original bacteria liquid into the other part, adding an equal amount of the filtered filtrate into the other part, and adding an equal amount of diluted 20-fold filtrate into the last part. Each seed was soaked for 30min with the top down.

Preparing a potato glucose agar culture medium, sterilizing at 121 ℃ and 0.1Mpa for 20min, inverting the culture medium, inoculating sclerotinia sclerotiorum on the potato glucose culture medium after the culture medium is cooled and solidified, culturing for 3-5d, inoculating the treated sunflower seeds into the culture medium after hyphae overgrow, and inoculating 10 sunflower seeds in each dish.

After the sunflower seeds are cultured for 2d, the morbidity condition of the seed hulls is checked, the record is carried out, the hulls are overturned by using tweezers, and the condition of the fungal hyphae on the hulls of the contact part of the culture medium and the sunflower hulls is observed according to three different levels. These three levels are as follows: level 0: the sunflower is antibacterial, and no hypha exists on the sunflower husk; level 1: the sunflower is slightly susceptible, and a small amount of hyphae can be seen on the sunflower husk; and 2, stage: the susceptible type is characterized in that hyphae are obviously grown on the sunflower hulls, and the results of an experimental group and a control group are recorded.

TABLE 2 influence of different treatment modes of ZX6 fermentation broth on sunflower seed hull infection

Note: the infection index is the number of infection times the number of infection stages; the incidence rate is divided by the number of infected diseases and the total number is multiplied by 100%.

Second, sunflower seed germination experiment

The ability of strain ZX6 to inhibit Sclerotinia sclerotiorum was investigated by seed germination. The control experiment was used to analyze the effect of the hyphae of strain ZX6 and its fermentation products on sunflower seed germination. The ZX6 bacterial liquid and sunflower seed treatment method adopts ZX6 bacterial liquid and seed treatment method in sunflower husk experiment, then each seed is soaked for 30min upside down, finally the sunflower seeds are respectively added into corresponding culture dishes added with filter paper, and a proper amount of sterile water is added to the surface of each seed, so that the seeds can germinate better.

Finally, the five seeds were placed in a constant temperature incubator at 28 ℃. And observing the germination condition of the seeds every 12 h. The results of the effect of ZX6 inoculum on seed germination were recorded (FIG. 4).

Thirdly, bacterial strain ZX6 in vitro leaf prevention effect rescreening

The strain ZX6 is inoculated into 100mL of Gao's No. one liquid culture medium and cultured for 24h at the temperature of 28 ℃ and at the speed of 170 r/min. The cell fermentation broth was diluted 20 times to obtain a treated solution. Selecting true leaves of sunflower with consistent leaf age, washing with sterile water, soaking in the treatment solution for 10min, air drying (no water drop on the surface), inoculating growing Helianthus annuus sclerotinia sclerotiorum mycelium blocks (diameter 6mm), inoculating 1 mycelium block (20 pieces each for treatment, repeating for 3 times) for each leaf, placing potato glucose agar culture medium blocks with diameter 6mm as blank control on water agar culture medium, culturing at constant temperature of 25 deg.C, and observing the disease spot expansion conditions of the experimental group (figure 5) and the control group (figure 6) after 24 h.

Fourth, sunflower potting experiment

Planting five-pot sunflowers, adding a proper amount of sunflowers seeds into each nutrition pot, placing the nutrition pots in a place with sufficient sunlight, ensuring that the sunflowers can normally sprout and grow seedling plants, watering the sunflowers every 1d, and carrying out subsequent experiments until the cultured sunflowers can grow two pairs of true leaves.

Adding the zymogen liquid of sclerotinia sclerotiorum obtained in the experiment into a spray can, and spraying 100ml of each of the roots of the sunflower seedlings of the experiment group, the blank control group and the experiment control group to ensure that other plants cannot be influenced when spraying. Experimental groups: spraying 100ml of sclerotinia sclerotiorum original bacteria liquid and then spraying 100ml of zymogen bacteria liquid of a strain ZX6 to serve as an experimental group; blank control group: spraying 100ml of sclerotinia sclerotiorum original bacteria liquid and then spraying 100ml of sterile water as a blank control; experimental control group: spraying 100ml of sclerotinia sclerotiorum original bacteria liquid, then spraying 100ml of potato dextrose agar liquid culture medium as an experimental control group, and recording the experimental results of each group (fig. 7 is a zymogen original bacteria liquid experimental group, fig. 8 is a blank control group, and fig. 9 is an experimental control group).

From the comparison of the results in fig. 7, fig. 8 and fig. 9, it is obvious that the zymogen liquid using the strain ZX6 can effectively inhibit the infection of sclerotinia sclerotiorum to the roots of sunflower, play a role in sterilization and promote the growth of sunflower.

Claims

1. Streptomyces cinnamoneumochromogenes (I) for preventing and treating sunflower sclerotiniaStrepomycescinnamonensis) ZX6 with preservation number of CGMCC 13116.

2. The Streptomyces cinnamoochromogenes (C) according to claim 1Strepomycescinnamonensis) Use of ZX6, characterized in that: the strain ZX6 is used for preventing and treating sunflower sclerotinia rot.