CN107058178B

CN107058178B - Streptomyces flavetris, microbial inoculum and application thereof

Info

Publication number: CN107058178B
Application number: CN201710198170.9A
Authority: CN
Inventors: 薛泉宏; 李玉龙; 来航线
Original assignee: Shaanxi Botai Bioengineering Co ltd; Northwest A&F University
Current assignee: Shaanxi Botai Bioengineering Co ltd; Northwest A&F University
Priority date: 2017-03-13
Filing date: 2017-03-29
Publication date: 2020-08-04
Anticipated expiration: 2037-03-29
Also published as: CN107058178A

Abstract

The invention specifically discloses a Streptomyces flaviviridis (Streptomyces flavivicini) 25, which is preserved in China Center for Type Culture Collection (CCTCC) at 2016, 3, 14 days, with the preservation number of CCTCC NO: m2016121. The active ingredients of the streptomyces flaviviridis microbial inoculum are live streptomyces flaviviridis spores and/or secondary metabolites of streptomyces flaviviridis. Also discloses the application of the streptomyces flavotrivialis and the microbial inoculum thereof in preventing and treating southern blight. The streptomyces flavus of the invention can produce active substances for resisting sclerotium rolfsii, can dissolve the cell wall of the sclerotium rolfsii, and simultaneously produce enzyme-soluble antibacterial action and active substance antagonistic action on pathogenic hyphae. The xanthothricin streptomycete applied to unsterilized soil can obviously promote the decay of sclerotium rolfsii in the soil, reduce the germination activity of the non-decayed sclerotium, reduce the pathogenic quantity from the source and reduce the harm of southern blight.

Description

Streptomyces flavetris, microbial inoculum and application thereof

Technical Field

The invention relates to the field of biological control of plant diseases, in particular to streptomyces flavetris, a microbial inoculum and application thereof.

Background

Southern blight is a common plant soil-borne fungal disease caused by Sclerotium rolfsii (Sclerotium rolfsii), mainly occurs at the stem base of crops, and when the soil is wet, a white hypha layer is formed on the surface of a diseased plant and the surrounding soil, and then Sclerotium shaped like rapeseeds develops. In the later period of disease, the rot of the base of the stem causes the obstruction of water and nutrition transportation, and the crop is withered and dead. The sclerotium rolfsii hosts exceed 500 species, and the damage to agricultural production is great. Sclerotium rolfsii can generate sclerotium, so that the resistance of pathogenic bacteria to pesticides and other control measures is improved, and the control difficulty of southern blight is increased. Because the residue of chemical pesticide in agricultural products and environment limits the use of the chemical pesticide in a large amount, the methods of crop rotation, soil disinfection and the like have poor sclerotium eliminating effect, and the exploration of a new prevention and control approach is a problem to be solved at present. Biological control can avoid the problems of food and environmental risks caused by chemical pesticides, and has attracted extensive attention.

Currently, biological control of southern blight is mainly focused on trichoderma and bacteria, few reports of inhibiting sclerotinia rolfsii (Sclerotium rolfsii) by utilizing actinomycetes are reported, and no report of forming Sclerotium rolfsii and inhibiting Sclerotium germination in soil by aligning actinomycetes secondary metabolites and viable bacteria preparations with Sclerotium rolfsii is reported.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide the streptomyces flaviviridis, the microbial inoculum and the application thereof, so as to effectively inhibit sclerotium rolfsii and prevent and control southern blight and similar plant diseases.

In order to achieve the purpose, the invention is realized by adopting the following technical scheme.

The Streptomyces flaviperidae is specifically Streptomyces flaviperidii (Streptomyces flaviperidii) 25 which is preserved in China center for type culture collection 3 and 14 days 2016, wherein the preservation unit address is Wuchang Lodojia mountain Wuhan university in Wuhan city, Hubei province, the code 730072, the preservation number is CCTCC NO: m2016121.

The Streptomyces flaviviridis (Streptomyces flavivicini) 25 is sequenced, and the 16S rDNA sequence is shown as SEQ ID No. 1.

The Streptomyces flavus microbial inoculum is characterized in that the effective components of the Streptomyces flavus microbial inoculum are Streptomyces flavus (Streptomyces flavitrini) 25 viable bacteria spores and/or secondary metabolites of the Streptomyces flavus (Streptomyces flavitrini) 25.

Preferably, the microbial inoculum is a powdery microbial inoculum, and the viable count is 3.3 × 10¹¹cfu/g。

Application of streptomyces flavus in preventing and treating plant diseases.

And (V) application of streptomyces flavus in preventing and treating southern blight.

(VI) the application of the streptomyces flavus microbial inoculum in preventing and treating plant diseases.

And (seventhly) application of the streptomyces xanthotrizanioides microbial inoculum in preventing and treating southern blight.

Compared with the prior art, the invention has the beneficial effects that:

streptomyces flaviviridis can synthesize chitinase, glucanase and cellulase, and can simultaneously generate active substances for resisting sclerotinia rolfsii. These fungal cell wall hydrolases are capable of solubilizing the cell walls of sclerotium rolfsii and at the same time producing an enzymatic antibacterial action and an active substance antagonistic action on pathogenic hyphae. The powdery viable bacteria preparation of the streptomyces flavotricin can obviously promote the decay of sclerotium rolfsii in soil when being applied to unsterilized soil, obviously reduce the germination activity of the non-decayed sclerotium, reduce the pathogenic quantity from the source and lighten southern blight and similar plant diseases.

Drawings

The invention is described in further detail below with reference to the figures and specific embodiments.

FIG. 1 is a characteristic diagram of a colony of Streptomyces flaviviridae 25;

FIG. 2 is a phylogenetic tree of Streptomyces flavotrivialis 25;

FIG. 3 is a graph showing the effect of inhibiting formation of sclerotium rolfsii by actinomycete cell-free fermentation filtrate, wherein: a shows the sclerotium formation amount of sclerotium rolfsii on a control CK culture medium; b is a chart of the effect of leveling the number of sclerotium rolfsii and the inhibition effect on sclerotium formation when the streptomyces flavacinus cell-free fermentation filtrate is diluted by 5 times; c is a map of the number of sclerotium rolfsii when the streptomyces flavus cell-free fermentation filtrate is diluted by 25 times and the inhibition effect on sclerotium formation, d is a map of the number of sclerotium rolfsii when the streptomyces flavus cell-free fermentation filtrate is diluted by 50 times and the inhibition effect on sclerotium formation;

FIG. 4 is a graph showing the effect of adding PDA medium to actinomycete acellular fermentation filtrate on sclerotium germination, wherein a is a graph showing the conditions of sclerotium germination and hyphae growth on a CK medium of a control group, and b is a graph showing the effect of inhibiting sclerotium germination on a PDA medium to which Streptomyces flavus acellular fermentation filtrate is added;

FIG. 5 is a graph showing the effect of the enzyme-induced teratogenesis of the Streptomyces flavedo cell-free fermentation filtrate on pathogenic hyphae, wherein a is the effect of the control CK fermentation filtrate treatment on the pathogenic hyphae, and b is the effect of the enzyme-induced teratogenesis of the Streptomyces flavedo cell-free fermentation broth treatment on the pathogenic hyphae.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention.

The invention provides a Streptomyces flaviviridis (Streptomyces flavotricini)25 for antagonizing southern blight, which is separated from root zone soil of konjaku southern blight in satan agagazel county, and the colony characteristics are shown in figure 1.

16S rDNA sequence analysis was performed on Streptomyces flavivicini 25, the phylogenetic tree of which is shown in FIG. 2, and the nucleotide sequences are as follows:

CTGCAGTGTCTGGGATTGCCATTCTTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTTCACTCTGGGACAAGCCCATGGAAACGGGGTCTAATACCGGATACGACTGCGGAAGGCATCTTCTGCGGTGGAAAGCTCCGGCGGTGAAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGATGTGAAAGCCCGAGGCTTAACCTCGGGTCTGCATTCGATACGGGCTAGCTAGAGTGTGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGGAACTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATATACCGGAAAGCATTAGAGATAGTGCCCCCCTTGTGGTCGGTATACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTGTGTTGCCAGCATGCCCTTCGGGGTGATGGGGACTCACAGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGTGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAAT CGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCTCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCTTGTGAGGGAGCGGTCGAAGCTCTACTCTGC

the test materials used in this example and their preparation were as follows:

the pathogenic bacteria to be tested in this example were Sclerotinia sclerotiorum (Sclerotium rolfsii), and aconitum carmichaeli which was infected by southern blight in the field of Nanzheng county, Shanxi province was isolated and identified by the institute of microbial resources, resource Environment institute of university of agriculture and forestry, northwest.

The Streptomyces flaviviridae (Streptomyces flavotricini)25 of the present example is a screening and identification collection of the microorganism resource research institute of resource environmental institute of northwest university of agriculture and forestry.

The soil of the embodiment is collected from a large cultivation layer of monkshood with southern blight, dried in air, crushed and screened by a 2mm sieve, and is used for determining the inhibition of the viable actinomycete preparation on sclerotium germination in the soil.

Preparation of Streptomyces flaviviridis preparation, solid fermentation of Streptomyces flaviviridis is carried out to prepare spore powder, the viable count of the obtained bacterial agent is 3.3 × 10¹¹cfu/g, the effective components of the streptomyces flaviviridis 25 viable bacteria spore and the secondary metabolite of the streptomyces flaviviridis 25.

The preparation method of sclerotium rolfsii powder comprises picking appropriate amount of activated hypha of sclerotium rolfsii with PDA slant with bamboo stick, inoculating into 500m L shake flask containing 200m L Cnahs' liquid culture medium, and culturing at 25 deg.C for 160r min^-1Shaking and culturing for 5 d. Filtering with double-layer gauze, collecting mycelium, washing with deionized water for several times until no residue is left, oven drying at 40 deg.C, grinding into fine powder with mortar, and sieving with 0.25mm sieve.

The preparation of target bacteria pathogenic bacteria cake comprises picking mycelium of Sclerotinia sclerotiorum (Sclerotiumrolfsii) cultured in dish for 3 days, adding into test tube containing 3m L sterile water and fine quartz sand, grinding with sterile glass rod to obtain mycelium segment suspension, sucking 0.05m L on PDA culture medium, spreading uniformly with spatula, culturing at 28 deg.C for 4 days, and making into circular cake with diameter of 7mm with sterile puncher.

Preparation of sclerotium: picking up the hypha of the sclerotium rolfsii growing on the inclined plane of the PDA to a PDA plate, culturing for 3 weeks at 28 ℃, scraping off the sclerotium from the plate, and sucking water by using a sterilized absorbent paper for later use.

A streptomyces flavus cell-free fermentation filtrate taking starch as a carbon source is prepared by adding 3m L sterile water to an actinomycete streptomyces flavus slant which is cultured to grow spores, scraping the spores off by using a sterilized bamboo stick, uniformly stirring to prepare a spore suspension, transferring the spore suspension into a 250m L triangular flask which is sterilized and is filled with 100m L Gao's No.1 liquid culture medium by using a sterile straw, sealing by using 4 layers of cotton cloth, repeating the steps of 3 bottles, carrying out shake culture at 28 ℃ and 160r/min for 8 days, filtering by using filter paper, and filtering and sterilizing by using a 0.22 mu m sterilized microporous filter membrane, thus obtaining the streptomyces flavus cell-free fermentation filtrate taking starch as the carbon source.

A process for preparing the liquid containing xanthotrienin hydrolase includes such steps as using the powder of sclerotium rolfsii as carbon source to replace starch in the liquid culture medium No.1, sterilizing at 121 deg.C for 30min, culturing at 28 deg.C and 160r/min for 5 days, standing, taking 20ml of supernatant, centrifugal culturing at 10000r/min for 5min, filtering the supernatant with 0.22 microns of millipore filter membrane, and sterilizing.

Example 1

Inhibition of hyphal growth by Streptomyces flavotricini 25

(1) The test method comprises the following steps:

mixing the Streptomyces flavacinus cell-free fermentation filtrate (stock solution, 5-10 times of sterile water dilution) and PDA culture medium cooled to about 50 deg.C at volume ratio of 1: 4, and pouring into flat plate to obtain PDA flat plate of fermentation filtrate diluted to 5-25-50 times; picking out Sclerotium rolfsii (Sclerotium rolfsii) agar block in the center of the plate with the bacteria face upward by using a sterilized bamboo stick, and repeating the treatment for 3 times; sterile water was used as a control. Culturing at 28 deg.C, measuring colony diameter by cross method after 24h and 48h, respectively, and calculating the antibacterial Rate of cell-free fermentation filtrate on hypha Growth according to the following formula (Inhibition Rate of Mycelia Growth, IR)_MG)

In the formula: IR_MGThe antibacterial rate of antagonistic bacteria cell-free fermentation filtrate on the growth of hyphae D_CK、D_tControl and treated colony diameters were obtained.

(2) And (3) test results:

as can be seen from Table 1, when starch and the sclerotium rolfsii mycelia are used as carbon sources, Streptomyces flaviviridae can produce active metabolites that can inhibit the growth of the sclerotium rolfsii mycelia. When starch is used as a carbon source, when cell-free fermentation liquor of streptomyces flaviviridis is diluted by 5, 25 and 50 times, the bacteriostasis rates of 48h of culture are respectively 52.8%, 36.1% and 25.0%, the bacteriostasis rates of 72h of culture are respectively 46.1%, 28.4% and 17.6%, and the bacteriostasis rates show the trend of gradually reducing along with the increase of dilution and the prolonging of culture time; when the sclerotium rolfsii thalli is used as a carbon source, the streptomyces flaviviridis can also generate active substances for inhibiting the growth of the sclerotium rolfsii hyphae, but the bacteriostasis rate of the streptomyces flaviviridis cell-free fermentation liquor at the same time and the same dilution is slightly lower than that of the streptomyces flaviviridis cell-free fermentation filtrate when starch is used as the carbon source.

TABLE 1 inhibition rate (%) -of Streptomyces flaviviridae cell-free fermentation filtrate aligned to the growth of whole sclerotium rolfsii hyphae

Example 2

Inhibition of sclerotia formation by Streptomyces flavotrichum (Streptomyces flavotricini)25

(1) The test method comprises the following steps:

after the determination of the bacteriostatic Rate of the streptomyces flavetris on the growth of hyphae is completed, the culture dish is placed at room temperature for culturing for 20 days, the produced sclerotia is scraped and counted, and the Inhibition Rate of sclerotia formation (Inhibition Rate of SclectiaFormation, IR)_SF) Calculated as follows:

in the formula: IR_SFInhibition of sclerotia formation, N_CK、N_tThe number of sclerotia formed in the control and treatment dishes, respectively.

(2) Test results

As can be seen from Table 2 and FIG. 3, the fermentation broth of Streptomyces flavus with different dilution times all had inhibitory effect (P < 0.01) on sclerotium formation of Sclerotinia sclerotiorum when cultured for 20 days. The inhibition effect of the streptomyces flaviviridis is good, when the streptomyces flaviviridis is diluted by 5 times, 25 times and 50 times, the produced sclerotia number is respectively 5/dish, 234/dish and 327/dish, and the inhibition rates of the antibacterial active substance in the cell-free filtrate on sclerotia formation are respectively 99.5%, 79.8% and 71.8%. The ability of the Streptomyces flaviviridis fermentation broth to inhibit sclerotia formation decreases with increasing dilution factor.

TABLE 2 inhibition of Streptomyces flaviviridae cell-free fermentation filtrate to sclerotium rolfsii sclerotium formation

Note that different capital letters after the same column of data indicate significant differences between different treatments (P < 0.01) as tested by the L SD method.

Example 3

Inhibition of sclerotium germination by Streptomyces flavotrichum (Streptomyces flavotricini)25

(1) Test method

(1.1) directly adding the Streptomyces flaviviridis cell-free fermentation filtrate into a culture medium to inhibit the germination of sclerotia:

uniformly mixing a Streptomyces xanthus cell-free fermentation filtrate stock solution and a PDA culture medium cooled to about 50 ℃ in a volume ratio of 1: 4, pouring the mixture into a flat plate, inoculating sclerotia on the PDA flat plate by using an inoculating needle after the culture medium is solidified, inoculating 12 sclerotia on each dish, placing the plate in a dark environment, culturing at 28 ℃, observing the Germination condition for 1 time every 24 hours, and calculating the Inhibition Rate of the Streptomyces xanthus cell-free fermentation filtrate on sclerotia Germination of Sclerotium according to the following formula (IR)_SG)

In the formula: IR_SGThe inhibition rate of the fermentation liquor on sclerotium germination, N_CK、N_tThe number of the germination sclerotium in the control dish and the treatment dish is respectively.

(1.2) inhibition rate of Streptomyces flaviviridis cell-free fermentation filtrate soaking bacteria core on sclerotium germination:

diluting Streptomyces flavotricin cell-free fermentation filtrate stock solution by 0, 10²、10³Adding sclerotium into cell-free fermentation filtrate of different dilutions, soaking for 24h, inoculating soaked sclerotium onto PDA plate with 12 grains/dish by inoculating needle, culturing at 28 deg.C in dark environment, observing germination condition every 24h for 1 time, and calculating cell-free fermentation filtrate soaking sclerotium according to the following formulaInhibition of Germination (Inhibition Rate of Sclerotium Germination, IR)_SG)

After 48h, the photographs were photographed, and the Image color analysis was performed using Image Pro Plus 6.0 to determine the hyphal germination Area pixel values (Hypa Area Pixels, HAP) of the treated and control, respectively. The Inhibition Rate of the cell-free fermentation filtrate on new hyphae formed by the germination of sclerotia (Inhibition Rate of Sub-Mycelia, IR) was calculated according to the following formula_SM)：

In the formula: IR_SMThe inhibition rate of the fermentation liquor after soaking on the growth of new hyphae generated by the germinating sclerotium, HAP_CKAnd HAP_tThe pixel values of the area of the new hyphae after the germination of the control sclerotium and the new hyphae after the germination of the treatment sclerotium are respectively shown.

(1.3) inhibition of xanthothrin streptomycete agent on sclerotium germination in soil:

according to the method of Rafik, etc., the streptomyces flavotrivialis viable bacteria agent is added into the air-dried soil of the aconite planting farmland layer according to the dosage of 0.5, 1.0, 5.0 and 10.0g/kg, and is fully and uniformly mixed, so that the spore concentration of the streptomyces flavotrivialis reaches 1.7 × 10⁸、3.4×10⁸、1.7×10⁹、3.3×10⁹cfu/g soil, then loading the soil inoculated with the streptomyces flavus viable bacteria preparation into a culture dish for two times according to the dosage of 50 g/dish: filling 30g of soil in each dish, strickling, uniformly placing sclerotia in 25 grains/dish, covering the rest soil on the sclerotia in 20 g/dish, adding 20ml of water in each dish, culturing at 28 ℃ for 14 days, and supplementing 2 times and 10ml of water in the culture period. After the culture is finished, picking out all sclerotia in soil in the dish, washing the soil on the surface of the sclerotia with tap water, then sterilizing the surface of the sclerotia for 3min with 2% sodium hypochlorite, washing the sclerotia with sterile water for 3 times, and then placing the sclerotia after surface sterilizationThe germination rate was observed after 3 days of incubation at 28 ℃ on PDA plates. The above treatment was repeated for 5 dishes per concentration. The Germination Inhibition Rate of the live Streptomyces flavotricin preparation on sclerotium in soil (IR) was calculated according to the following formula_F)。

In the formula: IR_FThe inhibition rate of the viable actinomycete preparation on the sclerotia germination in the soil, N_CK、N_tThe number of sclerotium germinates in the dish is respectively controlled and treated by adding the streptomyces flaviviridis viable bacteria preparation.

The effect of the microbial inoculum, namely the increase rate (delta CK) of microbial inoculum treatment compared with the control group is calculated according to the following formula

(2) Test results

(2.1) adding the Streptomyces flaviviridis cell-free fermentation filtrate into a culture medium to align the inhibition effect of sclerotium rolfsii sclerotium germination:

as can be seen from Table 3, when the sclerotium is placed on the PDA plate which is diluted by 5 times of the antagonistic bacteria and cultured for 24 hours, the inhibition rate of the streptomyces flavetrins to the sclerotium rolfsii germination reaches 100%. As the culture time is prolonged, the inhibition rate of the streptomyces flaviviridis on sclerotium germination is not influenced. As shown in FIG. 4, the inhibition rate of Streptomyces flaviviridis fermentation broth on sclerotium germination was still 100% at 96h of culture. According to later observations, the Streptomyces flaviviridae treated sclerotia had lost the ability to germinate completely.

TABLE 3 influence of Streptomyces flaviviridis cell-free fermentation filtrate diluted 5-fold with medium on sclerotium rolfsii sclerotium germination

(2.2) the inhibition effect of the Streptomyces flaviviridis cell-free fermentation filtrate soaking bacteria core on sclerotium germination:

as can be seen from Table 4, the fermentation broth of Streptomyces flavotricins has a certain inhibition effect on sclerotium germination when the sclerotium is soaked. When the culture is carried out for 24h and 48h, the inhibition rates of the antagonistic bacteria on sclerotium are 47.2 percent and 2.8 percent respectively, and the germination numbers of the sclerotium treated by the fermentation liquor with the rest dilution times have no obvious difference with the control, which indicates that the antagonistic bacteria cell-free fermentation filtrate can only delay the germination of the sclerotium and cannot completely lose the germination capacity. Nevertheless, the fermentation filtrate soaking still has significant inhibition effect on the growth of new hyphae generated after sclerotium germination, and the streptomyces flavidus fermentation filtrate stock solution is 10 times and 10 times as high as that of the streptomyces flavidus fermentation filtrate stock solution²The inhibition rates of the double dilution solution on the growth of new hyphae respectively reach 82.8%, 72.6% and 77.1%.

TABLE 4 inhibition rate of Streptomyces flaviviridis fermentation broth soaking treatment on sclerotium germination and new hypha growth of sclerotium rolfsii

Note that different capital letters after the same dilution gradient and the same column of data indicate that the cell-free fermentation filtrates of different strains align with the germination and growth inhibition rate of sclerotium rolfsii at the same time, and the difference is extremely obvious (P is less than 0.01) through the detection of L SD method.

(2.3) Effect of Streptomyces flaviviridis viable bacteria preparation on the activity of sclerotia in soil

As can be seen from Table 5, the addition of the Streptomyces flaviviridae viable bacteria preparation has a promoting effect on the decay of sclerotia in soil, but the germination rate of residual sclerotia is still high, when the dosage of the microbial inoculum reaches 10.0g/kg, the total germination rate of sclerotia is reduced to 44%, the inhibition effect on sclerotia germination is shown (P is less than 0.01), and the harm of southern blight is reduced from the source.

TABLE 5 influence of Streptomyces flaviviridis live bacterial preparations on decay and germination of sclerotia in field soil

Example 4

The activity of fungal cell wall hydrolase synthesized by Streptomyces flavotricini 25 and the enzymatic lysotropic teration of aligned sclerotium rolfsii hyphae.

(1) Test method

When the sclerotium rolfsii thallus is used as a carbon source, streptomyces flavetrics can be induced to generate fungal cell wall hydrolase, the cell-free fermentation filtrate is crude enzyme liquid of the induced enzyme, and the crude enzyme liquid is used as a material to measure the activity of the hydrolase of 3 fungal cell wall components.

(1.1) chitinase Activity assay:

preparation of chitin colloid and chitinase Activity measurement referring to Shanmugaiah et al method, 1m L of the prepared crude enzyme solution of Streptomyces flavatriensis inducible enzyme is added with 1m L10 g. L^-1The colloidal chitin is evenly mixed and then put into a constant temperature water bath with the temperature of 37 ℃ for hydrolysis for 3h, the mixture is boiled in a boiling water bath for 5min to terminate the reaction, deionized water is used for fixing the volume to 3ml, and the volume is controlled at 4000 r.min^-1Centrifuging for 5min, collecting supernatant of 1m L, and determining N-acetylglucosamine content in the hydrolysate by DNS method.

(1.2) β -1, 3-glucanase Activity assay:

referring to the Noronha et al method, 0.5m of crude enzyme solution prepared from L.1.2.2 was added with 0.5m of L10 g. L^-1The laminarin is mixed evenly and then put into a constant temperature water bath with the temperature of 45 ℃ for hydrolysis for 30min, the chitinase is subjected to subsequent treatment, and 1m L hydrolysate is taken to determine the glucose content in the hydrolysate by a DNS method.

(1.3) Filter paper cellulase activity assay:

referring to Ghose et al, 1m L0.05.05 mol L was added to a crude enzyme solution prepared at 0.5m L1.2.2^-1The citric acid buffer solution with the pH value of 4.8 is evenly mixed and put into 1 Xinhua filter paper strip of 1 × 6cm, and the Xinhua filter paper strip is put into a thermostatic water bath with the temperature of 50 ℃ for hydrolysis for 60min, and the subsequent treatment and the glucose determination are the same as β -1, 3-glucanase.

The above 3 enzyme assays were performed in the same manner as the high temperature inactivated enzyme solution, and were repeated 3 times per treatment, wherein the enzyme activity was defined as 1U for chitinase, which produces 1. mu. g N-acetylglucosamine by hydrolyzing chitin in 1min with 1m L enzyme solution at 37 ℃, 1U for β -1, 3-glucanase, which produces 1. mu.g of glucose by hydrolyzing laminarin in 1min with 1m L enzyme solution at 45 ℃, and 1U for filter cellulase, which produces 1. mu.g of glucose by hydrolyzing filter paper in 1min with 1m L enzyme solution at 50 ℃.

(1.4) enzymatic teratogenicity that fungal cell wall hydrolase synthesized by Streptomyces flaviviridae aligns to sclerotinia rolfsii hyphae:

inserting the cleaned and sterilized cover glass into a PDA (personal digital assistant) plate connected with sclerotium rolfsii, after hyphae are fully paved on the cover glass, inclining the cover glass fully paved with the hyphae against the wall of an upright 50m L sterilized centrifugal tube, adding a streptomyces flaviviridis cell-free fermentation filtrate into the centrifugal tube until the height of the cover glass is about 2/3, preserving the heat at 37 ℃ for 48 hours, taking out the cover glass, wiping off the hyphae on one surface of the cover glass, drying the cover glass in the air, observing the difference between the hyphae immersed and the hyphae not immersed in the cover glass under an optical microscope, checking whether the fermentation filtrate has a dissolving effect on the hyphae, and taking a picture.

(2) Test results

(2.1) fungal cell wall hydrolase synthesized by Streptomyces flaviviridae:

as can be seen from Table 6, when the sclerotium rolfsii cells were used as a carbon source, Streptomyces flaviviridae could be induced to synthesize a hydrolase capable of degrading the cell wall components of fungi, and Streptomyces flaviviridae could simultaneously synthesize chitinase, β -1, 3-glucanase and 3 enzymes of filter paper cellulase.

TABLE 6 Activity of sclerotium rolfsii thallus induced cell wall degrading enzymes of 3 extracellular fungi in Streptomyces flaviviridae fermentation broth

(2.2) enzymatic teratogenic action:

FIG. 5 is a graph showing the enzymatic teratogenic effect of fermentation broth on pathogenic hyphae. As can be seen from FIG. 5, the CK in the control group is normal hypha of pathogenic bacteria, and has uniform thickness, smoothness and transparency; the hypha treated by the streptomyces flaviviridis fermentation liquor can be seen to be dissolved under the action of the fermentation liquor.

① it can be known from the above examples that the application of the powdery viable bacteria preparation of the streptomyces flavipes into the unsterilized soil can obviously promote the decay of sclerotium rolfsii in the soil and obviously reduce the germination activity of the unsterilized sclerotium, the number of decayed sclerotium of the streptomyces flavipes with different dosages is 1.5-4.5 times of the reference, the germination rate of the residual sclerotium is respectively reduced by 21.0% -42.0% compared with the reference, ② can induce the streptomyces flavipes to synthesize chitinase, glucanase and cellulase and simultaneously generate active substances for resisting sclerotium rolfsii, which means that the fungal cell wall hydrolase generated by the streptomyces flavipes can dissolve the sclerotium rolfsii cell wall and simultaneously generate the enzyme-dissolving antibacterial action and the active substance antagonistic action on the pathogenic bacteria.

Although the present invention has been described in detail in this specification with reference to specific embodiments and illustrative embodiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto based on the present invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

<110> university of agricultural and forestry science and technology in northwest of Shaanxi Boqin bioengineering GmbH

<120> Streptomyces flavetris, microbial inoculum and application thereof

<130>2017

<160>1

<170>PatentIn version 3.5

<210>1

<211>1396

<212>DNA

<213> Streptomyces flaviviridi (Streptomyces flavotricini)25

<400>1

ctgcagtgtc tgggattgcc attcttggcg aacgggtgag taacacgtgg gcaatctgcc 60

cttcactctg ggacaagccc atggaaacgg ggtctaatac cggatacgac tgcggaaggc 120

atcttctgcg gtggaaagct ccggcggtga aggatgagcc cgcggcctat cagcttgttg 180

gtggggtaat ggcctaccaa ggcgacgacg ggtagccggc ctgagagggc gaccggccac 240

actgggactg agacacggcc cagactccta cgggaggcag cagtggggaa tattgcacaa 300

tgggcgaaag cctgatgcag cgacgccgcg tgagggatga cggccttcgg gttgtaaacc 360

tctttcagca gggaagaagc gaaagtgacg gtacctgcag aagaagcgcc ggctaactac 420

gtgccagcag ccgcggtaat acgtagggcg caagcgttgt ccggaattat tgggcgtaaa 480

gagctcgtag gcggcttgtc acgtcggatg tgaaagcccg aggcttaacc tcgggtctgc 540

attcgatacg ggctagctag agtgtggtag gggagatcgg aattcctggt gtagcggtga 600

aatgcgcaga tatcaggagg aacaccggtg gcgaaggcgg atctctgggc cattactgac 660

gctgaggagc gaaagcgtgg ggagcgaaca ggattagata ccctggtagt ccacgccgta 720

aacgttggga actaggtgtt ggcgacattc cacgtcgtcg gtgccgcagc taacgcatta 780

agttccccgc ctggggagta cggccgcaag gctaaaactc aaaggaattg acgggggccc 840

gcacaagcgg cggagcatgt ggcttaattc gacgcaacgc gaagaacctt accaaggctt 900

gacatatacc ggaaagcatt agagatagtg ccccccttgt ggtcggtata caggtggtgc 960

atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1020

ttgtcctgtg ttgccagcat gcccttcggg gtgatgggga ctcacaggag accgccgggg 1080

tcaactcgga ggaaggtggg gacgacgtca agtcatcatg ccccttatgt cttgggctgc 1140

acacgtgcta caatggccgg tacaatgagc tgcgataccg tgaggtggag cgaatctcaa 1200

aaagccggtc tcagttcgga ttggggtctg caactcgacc ccatgaagtc ggagtcgcta 1260

gtaatcgcag atcagcattg ctgcggtgaa tacgttcccg ggccttgtac acaccgctcg 1320

tcacgtcacg aaagtcggta acacccgaag ccggtggccc aacccttgtg agggagcggt 1380

cgaagctcta ctctgc 1396

Claims

1. Streptomyces flavacinus, in particular Streptomyces flavacinus (Streptomyces flavotricini)25, is separated from root zone soil of Konjac southern blight in Sagaku county of Shanxi province; the culture is preserved in China center for type culture Collection in 2016, 3 months and 14 days, and the preservation number is CCTCC NO: m2016121.

2. The Streptomyces flavedo according to claim 1, wherein the 16S rDNA sequence of Streptomyces flavedo (Streptomyces flavedocini) 25 is shown as SEQ ID No. 1.

3. A Streptomyces flaviviridae bacterial agent characterized in that the effective components are the live bacterial spores and/or secondary metabolites of Streptomyces flaviviridae (Streptomyces flavivicini) 25 according to claim 1.

4. The Streptomyces flavotricin microbial inoculum according to claim 3, wherein the microbial inoculum is a powdery microbial inoculum, and the viable count of the powdery microbial inoculum is 3.3 × 10¹¹cfu/g。

5. Use of streptomyces flaviviridae according to claim 1 or 2 for the prevention and treatment of southern blight.

6. The use of the Streptomyces flaviviridae bacterial agent of claim 3 or 4 in the prevention and treatment of southern blight.