CN116536224A - Streptomyces salt house Y88 and application thereof - Google Patents

Streptomyces salt house Y88 and application thereof Download PDF

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CN116536224A
CN116536224A CN202310802004.0A CN202310802004A CN116536224A CN 116536224 A CN116536224 A CN 116536224A CN 202310802004 A CN202310802004 A CN 202310802004A CN 116536224 A CN116536224 A CN 116536224A
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fusarium
streptomyces
bacteria
oxysporum
cucumber
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郭岩彬
李晓彤
任静
徐巧林
张洒洒
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China Agricultural University
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Abstract

The invention discloses Streptomyces salt house Y88 and application thereof. The strain Y88 has strong inhibition effect on plant pathogenic fungi of Botrytis cinerea, rhizoctonia cerealis, rhizoctonia solani, alternaria solani, anthracnose, fusarium oxysporum, cotton verticillium wilt, apii Graveolentis, rhizoctonia solani and Fusarium pseudoverticillatum 11, has obvious inhibition effect on plant pathogenic bacteria of Rhizoctonia cerealis, rhizoctonia solani and Rhizoctonia solani 5, and has broad-spectrum antibacterial activity. The biological control method can be applied to biological control of wheat stem basal rot caused by fusarium pseudograminearum and cucumber root rot caused by fusarium oxysporum, obviously reduces the occurrence rate of diseases and has good application prospect.

Description

Streptomyces salt house Y88 and application thereof
Technical Field
The invention relates to the fields of microbiology, biotechnology and biological control, in particular to Streptomyces salinosus Y88 and application thereof.
Background
The wheat stem rot is caused by fusariumFusariumsp.), a worldwide important disease caused by various fungi, especially fusarium pseudograminearum, can occur from seed germination to maturity, causes browning or rot of the root or stem base of wheat, and causes death of plants when serious, is a common soil-borne disease in the wheat planting process, is a wheat disease which rapidly grows in recent years, and seriously affects the yield and quality of wheat (Rocha et al 2011). In addition, various mycotoxins can remain in plants and seeds infected with the wheat stem-based rot, which constitutes a serious threat to human and animal health.
In vegetable production, root rot is the most harmful worldwide soil-borne disease, and is also a common disease in cucumber production. The disease can be generated in the whole growth stage of plants, and the cucumber root rot has the characteristics of quick transmission, deep hazard and large economic loss.
Actinomycetes are the earliest discovered microorganism with biocontrol effect, and a plurality of actinomycetes have strong inhibition effect on plant pathogenic fungi and are widely applied to biological control of plant diseases, wherein streptomyces is a species of the genus streptomycesStreptomyces sp.) Is most studied. Researches show that almost all streptomyces can produce active substances with antibacterial action, such as antibiotics, etc., such as streptomycesStreptomyces kasugenesis) The produced aminoglycoside antibiotics Kasugamycinum inhibit protein biosynthesis, and can be applied to the prevention and treatment of rice blast (He Yawen and the like, 2022); the macrolide antibiotic Anthramycin in the metabolite of Streptomyces cnH365 is particularly effective against diseases caused by Bacillus anthracis (Hensler et al 2014); streptomyces sp under greenhouse conditionsStreptomyces polychromogenesUAE 1) can obviously inhibit diseases caused by fusarium solani, and reduce the disease index by 53.0 percent (Albloom et al 2021); streptomyces (Streptomyces sp.)Streptomyces puniceusL75) fermentation broth extractionThe crude extract is taken out to reduce the early blight of tomato by 98.0% (Hao et al 2019). At present, no related report of preventing and controlling wheat stem basal rot and cucumber root rot caused by fusarium by Streptomyces salt house is found.
Disclosure of Invention
The invention aims to provide Streptomyces salina Y88 with broad-spectrum antibacterial activity and application thereof, in particular to application of the Streptomyces salina Y88 in biological control of plant diseases such as wheat stem basal rot and cucumber root rot.
In order to achieve the object of the invention, in a first aspect, the invention provides a strain Y88 with a biocontrol effect, which is classified and named as Streptomyces salinosusStreptomyces sioyaensisThe strain is separated from the Ackersu region of the Uygur autonomous region of Xinjiang in 2021, and is preserved in China general microbiological culture Collection center (CGMCC), address: beijing, the North Chen West Lu No. 1, 3 national academy of sciences of China microbiological study, post code 100101, preservation number CGMCC No. 27483, and date 2023, 5 months and 30 days.
In a second aspect, the present invention provides a microbial inoculum comprising Streptomyces siosphaericus Y88.
In a third aspect, the present invention provides a biopesticide or biofertilizer prepared from Streptomyces siosphaerensis Y88 or a microbial inoculum thereof.
In a fourth aspect, the present invention provides a biocontrol or antibacterial agent prepared from Streptomyces salt house Y88.
In a fifth aspect, the invention provides any one of the following uses of Streptomyces salinospora Y88 or a microbial inoculum thereof:
1) For antagonizing phytopathogenic fungi;
2) For antagonizing phytopathogenic bacteria;
3) Is used for preventing and treating wheat stem basal rot;
4) Is used for preventing and treating cucumber root rot;
5) Is used for preparing biological pesticides.
Plants of the invention include, but are not limited to, wheat, cucumber.
The plant pathogenic fungi of the invention are not limited to Fusarium pseudograminearumFusarium pseudograminearum) Fusarium oxysporum (F.oxysporum)Fusarium oxysporum) Fusarium moniliforme (F. Moniliforme)Fusarium moniliforme) Botrytis cinereaBotrytis cinerea) Rhizoctonia cerealis @Rhizoctonia cerealis) Rhinocerotis microorumCeratocystis fimbriata) Rhizoctonia solani of riceRhizoctonia solani) Tomato early blight germAlternaria solani) Lily root rot fungusFusarium oxysporum f.sp.lilii) Watermelon fusarium wiltFusarium oxysporum f.sp.niveum) Cotton fusarium wilt bacteriaVerticillium dahliae Kleb) Fusarium species of the round branchFusarium rotundiformis) Bacterial plaque of celerySeptoria apiicola Speg) The colletotrichum gloeosporioides isColletotrichum destructivum）。
The plant pathogenic bacteria of the invention are not limited to cucumber angular leaf spot bacteriaPseudomonas syringaepv.lachrymans) Citrus canker pathogenXanthomonas citri) Black rot of chinese cabbageXanthomonas campestrispv.campestris) Chilli scab germXanthomonas campestris pv.vesicatoria) Bacterial wilt of eggplantRalstonia solanacearum）。
In a sixth aspect, the present invention provides any one of the following uses of Streptomyces salinospora Y88 and/or bacteriostatic active substances produced thereof:
a) For combating pathogenic bacteria and related diseases or plant diseases caused by pathogenic bacteria;
b) For inducing plants to enhance resistance to related diseases or plant diseases caused by pathogenic bacteria;
c) Is used for preparing biocontrol agents, antibacterial agents or plant immunity inducing agents.
Further, the pathogenic bacteria include plant pathogenic fungi and plant pathogenic bacteria.
Further, the plant diseases include, but are not limited to, wheat stem rot and cucumber root rot.
The Streptomyces salt house Y88 has strong inhibition effect on 14 pathogenic fungi and 5 pathogenic bacteria, has a very wide antibacterial spectrum, can play a role in biocontrol in the plant growth process, and can also remarkably reduce the incidence and disease index of the root rot of the cucumber caused by wheat stem basal rot and fusarium oxysporum caused by fusarium pseudograminearum and fusarium moniliforme. The strain Y88 has good application value in the aspects of biological control of plant diseases and the like.
The inhibition rates of the strain Y88 on the fusarium pseudograminearum, the fusarium oxysporum and the fusarium moniliforme are 68.4 percent, 78.6 percent and 65.6 percent respectively; the plant antibacterial agent has obvious inhibition effects on the growth of the pathogenic bacteria of the eleven pathogenic bacteria of the gray mold bacteria, the tomato early blight bacteria, the small-length coral mold bacteria, the lily root rot bacteria, the wheat sheath blight bacteria, the rice sheath blight bacteria, the cotton verticillium wilt bacteria, the celery spot blight bacteria, the watermelon fusarium wilt bacteria, the verticillium wilt-like fusarium and the anthracnose bacteria, and has the broad-spectrum antibacterial activity, wherein the antibacterial rate of the pathogenic bacteria of the eleven pathogenic bacteria of the citrus canker bacteria, the eggplant bacterial wilt bacteria, the cucumber angular leaf spot bacteria, the pepper scab bacteria and the celery cabbage black rot bacteria is 52.79 mm, 35.54 mm, 41.72 mm, 38.26 mm and 19.39 mm. Greenhouse experiments prove that the prevention and treatment effect of the Y88 fermentation liquor on the wheat stem basal rot caused by the fusarium pseudograminearum reaches 65.0 percent; the control effect on cucumber root rot caused by fusarium oxysporum and fusarium moniliforme reaches 48.9 percent and 26.7 percent respectively. Streptomyces salt house Y88 has very good application prospect in the aspects of biological pesticides and antibacterial agents.
Drawings
FIG. 1A shows the inhibition effect of different strains on Fusarium pseudograminearum in a preferred embodiment of the present invention.
FIG. 1B shows the inhibition effect of different strains on Fusarium oxysporum in a preferred embodiment of the present invention.
FIG. 1C shows the inhibition effect of different strains on Fusarium moniliforme plates in a preferred embodiment of the present invention.
FIG. 2A shows the expansion of 8 strains of high F.pseudograminearum antibacterial material in a preferred embodiment of the present invention.
FIG. 2B shows the expansion capacity of 9 strains of highly anti-Fusarium oxysporum antibacterial material in a preferred embodiment of the present invention.
FIG. 2C shows the expansion of 6 strains of high resistance Fusarium moniliforme antibacterial material in a preferred embodiment of the present invention.
FIG. 3 is a phylogenetic tree of the 16S rRNA genes of the Y88 strain in the preferred embodiment of the present invention.
FIG. 4 is a phylogenetic tree of the tandem genes of strain Y88 in a preferred embodiment of the invention.
FIG. 5 shows morphological characteristics of strain Y88 on different media according to a preferred embodiment of the invention.
FIG. 6 shows the mycelium morphology of strain Y88 in the preferred embodiment of the present invention.
FIG. 7 shows the physiological and biochemical identification of strain Y88 in a preferred embodiment of the invention.
FIG. 8 shows the growth inhibitory effect of strain Y88 on 11 plant pathogenic fungi in a preferred embodiment of the invention.
FIG. 9 shows the growth inhibitory effect of strain Y88 on 9 plant pathogenic fungi in a preferred embodiment of the invention.
FIG. 10 shows the control effect of strain Y88 on wheat stem basal rot in the preferred embodiment of the invention. CK: wheat is planted in germ-free soil (clear water); fp: wheat is planted in diseased soil containing fusarium pseudograminearum; soaking wheat in Y88 fermentation broth, and planting in disease soil containing Fusarium pseudograminearum; carbendazim+fp: wheat is planted in disease soil containing fusarium pseudograminearum after seed soaking by using carbendazim.
FIG. 11 shows the control effect of strain Y88 on cucumber root rot caused by Fusarium oxysporum in a preferred embodiment of the present invention. CK: cucumber is planted in germ-free soil (clear water); fo: cucumber is planted in disease soil containing fusarium oxysporum; soaking cucumber seeds with Y88 fermentation broth and planting the cucumber seeds in disease soil containing fusarium oxysporum; carbendazim + Fo: the cucumber is planted in disease soil containing fusarium oxysporum after seed soaking by carbendazim.
FIG. 12 shows the control effect of strain Y88 on cucumber root rot caused by Fusarium moniliforme in the preferred embodiment of the present invention. CK: cucumber is planted in germ-free soil (clear water); fm: cucumber is planted in disease soil containing fusarium moniliforme; soaking cucumber seeds in Y88 fermentation broth and then planting the cucumber seeds in disease soil containing fusarium moniliforme; carbendazim+fm: the cucumber is planted in disease soil containing fusarium moniliforme after seed soaking by carbendazim.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.
The test bacteria referred to in the following examples were all derived from the university of agriculture in China as follows:
plant pathogenic fungi: fusarium pseudograminearumFusarium pseudograminearum) Fusarium oxysporum (F.oxysporum)Fusarium oxysporum) Fusarium moniliforme (F. Moniliforme)Fusarium moniliforme) Botrytis cinereaBotrytis cinerea) Rhizoctonia cerealis @Rhizoctonia cerealis) Rhinocerotis microorumCeratocystis fimbriata) Rhizoctonia solani of riceRhizoctonia solani) Tomato early blight germAlternaria solani) Lily root rot fungusFusarium oxysporum f.sp.lilii) Watermelon fusarium wiltFusarium oxysporum f.sp.niveum) Cotton fusarium wilt bacteriaVerticillium dahliae Kleb) Fusarium species of the round branchFusarium rotundiformis) Bacterial plaque of celerySeptoria apiicola Speg) The colletotrichum gloeosporioides isColletotrichum destructivum）。
Phytopathogenic bacteria: agrobacterium rhizogenes @Agrobacterium rhizogenes) Agrobacterium vitis @Agrobacterium vitis) Agrobacterium tumefaciens @Agrobacterium tumefaciens) Bacterial leaf spot of cucumberPseudomonas syringaepv.lachrymans) Citrus canker pathogenXanthomonas citri) Black rot of chinese cabbageXanthomonas campestrispv.campestris) Chilli scab germXanthomonas campestris pv.vesicatoria) Pectobacterium strain for soft rot of carrotPectobacterium carotovorum) Bacterial wilt of eggplantRalstonia solanacearum）。
Example 1 isolation screening and identification of Strain Y88
1. Isolation and screening of Strain Y88
Collecting soil sample in Akesu region of Uygur autonomous region of Xinjiang in 2021, weighing 10 g soil samples, adding into conical flask containing 100 mL sterile physiological saline, standing for 20 min, shaking with shaking table at 28deg.C and 150 rpm for 30 min, standing for 10 min, sucking 0.1 mL supernatant and 0.9 mL sterile physiological saline with a pipette, adding into 1 mL centrifuge tube, diluting, sequentially diluting for 10 min 2 ，10 3 ，10 4 Multiple, more than 100. Mu.L of the soil suspension was applied to a medium of Gao's first type (soluble starch 20 g, KNO 3 1 g，K 2 HPO 4 0.5 g，NaCl 0.5 g、MgSO 4 ·7H 2 O 0.5 g、FeSO 4 ·7H 2 O0.01-g and distilled water 1000 mL,pH7.2~7.4), culturing at 28deg.C for 5 days, picking single colony with different forms with sterile toothpick, streaking, purifying, and storing for use to obtain 15 actinomycetes.
2. Screening of highly antagonistic strains
Is prepared from Fusarium pseudograminearumFusarium pseudograminearum) Fusarium oxysporum (F.oxysporum)Fusarium oxysporum) Fusarium moniliforme (F. Moniliforme)Fusarium moniliforme) Three fusarium strains are used as target fungi, and a flat plate counter method is adopted to detect the growth inhibition effect of actinomycetes on plant pathogenic bacteria. Firstly, symmetrically inoculating the separated actinomycete strain at a position 2.5 and cm away from the center of a PDA plate, culturing for 5 days at 28 ℃, inoculating a pathogenic fungi bacterial cake with the diameter of 5 mm at the center of the plate, culturing at 22 ℃ until the control treatment grows on the whole culture dish by taking the PDA plate without actinomycete as a control, measuring the colony diameter of the pathogenic fungi, and calculating the inhibition ratio according to the formula I):
formula I
From 15 actinomycetes, 8 strains of fusarium pseudograminearum (figure 1A), 9 strains of fusarium oxysporum (figure 1B) and 6 strains of fusarium moniliforme (figure 1C) with stronger antibacterial activity (> 40.0%) are screened for a second time, and the expansion capacity of actinomycete antibacterial substances on a flat plate is measured: firstly, inoculating the separated actinomycetes on one side to the position 2.5 and cm away from the center of a PDA plate, culturing for 5 days at 28 ℃, inoculating a pathogenic fungi bacterial cake with the diameter of 5 mm on the center of the plate, taking the PDA plate without actinomycetes as a control, repeating each treatment for 5 times, culturing at 22 ℃ until the control treatment grows on the whole culture dish, and measuring the fan-shaped angle formed by pathogenic fungi.
After screening, finally selecting a strain Y88 with high antibacterial rate (40.0%) and strong antibacterial substance diffusivity for subsequent experiments, wherein the inhibition effect of the strain on the plates of three fusarium strains is shown in fig. 1A-1C and tables 1-1, 1-2 and 1-3, and the expansion capacity of the antibacterial substances is shown in fig. 2A-2C and tables 2-1, 2-2 and 2-3.
TABLE 1-1 growth inhibition of actinomycetes against Fusarium pseudograminearum
TABLE 1-2 growth inhibition of actinomycetes against Fusarium oxysporum
Tables 1-3 growth inhibition ratio of 15 actinomycetes to Fusarium moniliforme
Table 2-1 8 expansion ability of Fusarium pseudograminearum antibacterial substances
Table 2-2 9 expansion ability of Fusarium oxysporum antibacterial substance
Table 2-3 6 expansion ability of Fusarium moniliforme antibacterial substance
3. Identification of strains
Comprehensively considering morphological characteristics, physiological and biochemical indexes and 16S rRNA analysis results of the strain Y88, and identifying the strain as Streptomyces salinaStreptomyces sioyaensis. The method comprises the following steps:
(1) 16S rRNA sequence analysis
The bacterial genome rapid extraction kit is adopted to extract the genome DNA of actinomycetes, and the general primers 27F (5 '-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-CCCCGTCAATTCATTTGAGTTT-3') are used for amplifying the DNA, and the PCR reaction system and the amplification conditions are shown in tables 3-1 and 3-2. The sequence obtained after sequencing splice (SEQ ID NO: 1) was submitted to NCBI (National Center for Biotechnology Information, https:// www.ncbi.nlm.nih.gov /) data for alignment analysis and a phylogenetic tree of the 16S rRNA gene was constructed using the proximity ligation method (MEGA-X (Molecular Evolutionary Genetics Analysis) software). The results are shown in FIG. 3, strain Y88 andStreptomyces sioyaensis DSM 40032 was assigned to the same cluster with 99.5% homology.
TABLE 3-1 PCR amplification reaction System
TABLE 3-2 PCR amplification 16S rRNA reaction procedure
(2) Housekeeping gene sequence amplification and phylogenetic analysis
To further elucidate the relationship of the strain to related strains, the gene was selected for 5 housekeeping genesatpD、gyrB、recA、rpoB andtrpb was subjected to amplification sequencing (SEQ ID NO: 2-6), and the primers and amplification conditions are shown in Table 4-1 and Table 4-2. Will 5The sequence of housekeeping genes is sequenced from beginning to endatpD-gyrB-recA-rpoB-trpB are connected in series, and a phylogenetic tree is constructed by adopting a Neighbor-Joing method by using MEGA-X software. As a result, as shown in FIG. 4, housekeeping gene of strain Y88atpD、gyrB、recA、rpoB、trpThe similarity of B with its standard strain reaches 100%.
TABLE 4-1 PCR primers used in this experiment
TABLE 4-2 PCR amplification housekeeping Gene reaction conditions
(3) Morphological characterization of Y88 Strain
The strain was streaked and inoculated onto 10 different media such as ISP 1, ISP 2, ISP 3, ISP 4, ISP 5, ISP 6, ISP 7, NA, PDA and Nahniki solid medium, and cultured at constant temperature of 28℃for about 10 days, and when the colony appeared spores, the characteristics such as colony color, matrix mycelium color and growth condition were observed. Observing the mycelium morphology of the target strain by an inserting sheet method, obliquely inserting a sterile cover glass into a flat plate, inoculating a small amount of actinomycete strain spores to the base of one side of the cover glass, culturing at the constant temperature of 28 ℃ for 5 days, taking out, wiping off the culture medium on the back surface of the cover glass and the mycelium with poor growth, observing on the glass slide under a microscope, and taking a picture.
The results show that the colony morphology, the matrix mycelium color and the growth conditions on different culture media are different, the Y88 strain grows well on most culture media, the colony is white or light yellow (figure 5), the matrix mycelium is yellow, the mycelium is slender and has a large number of branches by microscopic observation, and small oval conidia are generated at different sites of the mycelium (figure 6).
(4) Physiological and biochemical characteristics of Y88 strain
Performing carbon source utilization, milk coagulation and peptonization, starch hydrolysis and nitrate reduction on Y88As shown in FIG. 7 and Table 5, 35 physiological and biochemical characteristics such as enzyme activity were tested, and the results of the measurement of the coagulation and peptonization of milk by the strain Y88, starch hydrolysis, catalase, oxidase, lipase Tween 20, lipase Tween 40, lipase Tween 80, and nitrate reduction were positive, and the results of the measurement of urease, casein hydrolysis, xylan hydrolysis, pectin hydrolysis, gelatin liquefaction, esculin hydrolysis, cellulose hydrolysis, and H were positive 2 S gives negative results, and arabinose, rhamnose, ribose, xylose, sucrose, raffinose, glucose, maltose, fructose, glycerol, inositol, mannitol, sorbitol, and citric acid, ribitol, and erythritol cannot be used. Can grow at 28 ℃ under the condition that the pH value is within the range of 5-10 and the NaCl content is 3.0-9.0%.
Salt tolerance, milk coagulation and peptonization, starch hydrolysis, esculin hydrolysis characteristics, and carbon source utilization characteristics and characteristics of raffinose, glucose, maltose, and the like of strain Y88Streptomyces sioyaensisThe standard strain is consistent, colony color is similar to that of the standard strain, but indexes such as temperature tolerance, gelatin liquefaction, urease, lipase and the like are different from those of the standard strain.
The 16S rRNA molecular identification result, phylogenetic tree, morphological characteristics and physiological and biochemical characteristics are combined, and the strain Y88 is identified as Streptomyces salinaStreptomyces sioyaensis）。
TABLE 5 physiological and biochemical test results of Strain Y88
Note that: "+" positive, "-" negative, "nd" indicates no data.
EXAMPLE 2 determination of broad-spectrum antibacterial Effect of Strain Y88
1. Inhibition of phytopathogenic fungi by strain Y88
The fungus bacteriostasis spectrum of the strain Y88 is detected by adopting a counter culture method, and the fungus bacteriostasis spectrum of the strain Y88 is respectively measured for gray mold fungusBotrytis cinerea) Tomato early blight germAlternaria solani) Rhinocerotis microorumCeratocystis fimbriata) Lily root rot fungusFusarium oxysporum f.sp.Lili) Watermelon fusarium wiltFusarium oxysporum f.sp.niveum) Rhizoctonia cerealis @Rhizoctonia cerealis) Rhizoctonia solani of riceRhizoctonia solani) Cotton verticillium wilt bacteriaVerticillium dahliae Kleb) Bacterial plaque of celerySeptoria apiicola Speg) Fusarium species of the round branchFusarium rotundiformis) The colletotrichum gloeosporioides isColletotrichum destructivum) 11 pathogenic fungi inhibiting effect. The assay is described in example 1.
The results show that Y88 has obvious inhibition effect on 11 plant pathogenic fungi, has broad-spectrum antibacterial activity (table 6 and figure 8), has the antibacterial rate exceeding 60.0% on botrytis cinerea, coral fungus, sheath blight germ of wheat, sheath blight germ of rice, early blight germ of tomato, anthracnose germ, watermelon fusarium wilt, cotton verticillium wilt and celery spot fusarium wilt, and is 79.9%, 79.0%, 79.5%, 71.2%, 76.7%, 79.0%, 73.4%, 66.4% and 73.2% respectively, and has lower antibacterial rates on lily root rot germ and fusarium wilt of round-shaped branch sample, which are 47.9% and 37.7% respectively. It is also speculated that the antibacterial substance produced by the Y88 strain may be an antibiotic substance, and that the substance is capable of antagonizing most pathogenic bacteria.
TABLE 6 inhibition of growth of phytopathogenic fungi by strain Y88
2. Inhibition of phytopathogenic bacteria by strain Y88
The bacterial inhibition spectrum of the strain Y88 is detected by a double-layer flat plate method, and the bacterial inhibition spectrum of the strain Y88 is respectively measured on agrobacterium rhizogenesAgrobacterium rhizogenesK27 Agrobacterium vitis @ aAgrobacterium vitisK308 Agrobacterium tumefaciens [ ]Agrobacterium tumefaciensC58 Bacterial leaf spot of cucumberPseudomonas syringaepv.lachrymans) Citrus canker pathogenXanthomonas citri) Chinese cabbage black rot germXanthomonas campestrispv.campestris) Chilli scabPathogenic bacteriaXanthomonas campestrispv.vesicatoria) Pectobacterium of carrot soft rotPectobacterium carotovorum) Eggplant bacterial wiltRalstonia solanacearum) 9 inhibition effect of plant pathogenic bacteria. Firstly, picking an activated actinomycete single colony, inoculating the actinomycete single colony to the center of a PDA flat plate, culturing for 5 days at 28 ℃, then taking 3 mL chloroform to a glass culture dish cover, and reversely fumigating for 10-12 h, so as to kill actinomycete thalli by steam. Picking single colony of plant pathogenic bacteria, inoculating in LB test tube, culturing at 28deg.C and 150 rpm for 8 h, regulating bacterial liquid OD with sterile physiological saline 600 After adding 100 μl of the bacterial suspension to 5 mL water agar medium cooled to about 50deg.C, mixing, immediately pouring the suspension onto chloroform steam inactivated actinomycetes PDA plate, spreading into uniform thin layer, continuously culturing at 28deg.C for 2 days, and measuring diameter of antibacterial circle.
As shown in Table 7 and FIG. 9, Y88 has obvious inhibition effect on the growth of pathogenic bacteria of plant pathogenic bacteria of citrus canker, eggplant bacterial wilt, cucumber angular leaf spot, capsicum scab, celery cabbage black rot 5, and the diameters of inhibition zones are 52.79 mm, 35.54 mm, 41.72 mm, 38.26 mm and 19.39 mm respectively, and have no inhibition effect on other 4 pathogenic bacteria of plant.
TABLE 7 inhibition of growth of phytopathogenic bacteria by Strain Y88
Example 3 greenhouse control of wheat Stem rot by Strain Y88
Preparing fungus soil: the pathogenic bacteria of the stem-based rot of the tested wheat is fusarium pseudograminearumFusarium pseudograminearumFp) was first inoculated to PDA liquid medium, cultured at 28℃for 5 days with shaking at 150 rpm, the liquid was inoculated to corn flour medium (corn flour 1000 g, distilled water 500 mL, mixed and steamed for 30 min, packed in vials, autoclaved at 121℃for 20 min) and incubated in an incubator at 28℃with slight shaking for the first two days to homogenize the growth of the mycelia. After the hypha grows to be full (about 10 d), the mycelium is taken out. Will grow up the germCorn flour medium and cultivation substrate (nutrient soil: vermiculite=1:1, volume ratio) according to 1:30, uniformly mixing the materials according to the mass ratio to prepare fungus soil;
preparation of strain Y88 fermentation broth: the Y88 strain after the activation is selected and inoculated into PDA liquid culture medium, and actinomycete bacterial liquid is prepared by shaking culture for 5 days at 28 ℃ and 150 rpm;
and (3) prevention effect determination: the wheat variety is Zhengmai 9023 (high-yield breeding laboratory of wheat institute of agricultural sciences of Henan province). Wheat seeds with uniform and full size and consistent white exposure are selected for control effect measurement, 3 treatments are set in the test, the wheat seeds are soaked in the strain Y88 fermentation liquor, 50% carbendazim wettable powder and sterile water for 3 h respectively, 10 seeds are treated each time, 6 pots are repeated, disease conditions are investigated twenty days after emergence, disease indexes and disease rates are investigated, and control effects are calculated.
The classification standard of the wheat stem rot disease condition is summarized as follows:
level 0: plants did not develop disease;
stage 1: brown stems in the ground or wheat first leaf sheath shows slight symptoms;
3 stages: the first leaf sheath turns brown, but the first leaf sheath does not turn black;
5 stages: the first leaf sheath turns black or the second leaf sheath turns brown;
7 stages: the third leaf sheath turns brown or the wheat grows slowly or nearly dies due to morbidity;
stage 9: wheat plants die from disease.
Incidence = (number of affected plants/total number of investigation) ×100%
Disease index=100×Σ (number of plants at each stage×relative number)/(total number of plants investigated×highest number of stages)
Relative control (%) = (control disease index-treatment disease index)/control disease index x 100%
By the method, the wheat seedlings are obviously affected by the stem basal rot caused by applying the falcate pseudograena in the soil, the stem basal part of the plant turns brown, the root is obviously shortened, the morbidity reaches 100.0%, and the disease index reaches 25.93. The morbidity and the disease index of the wheat stem basal rot can be obviously reduced by the strain Y88 and the carbendazim treatment group, the potting experimental result is shown in figure 10, the prevention effect of the inoculated strain Y88 fermentation liquor and the treatment of the carbendazim on the wheat stem basal rot can reach 65.0 percent, compared with a control, the morbidity of the wheat seedlings of the Y88 treatment group is reduced by 51.7 percent, the disease index is reduced by 9.1, the disease index is not obviously different from that of the 500 times 50 percent of the carbendazim treatment group, and the plant growth inhibition effect caused by the wheat stem basal rot can be reduced. The result shows that the antagonistic bacteria has an inhibiting effect on wheat stem basal rot caused by fusarium pseudograminearum and has good application potential.
Example 4 greenhouse control of cucumber root rot by Strain Y88
The pathogenic bacteria of the cucumber root rot to be tested is fusarium oxysporumFusarium oxysporumFo) and Fusarium moniliforme in shortFusarium moniliformeAbbreviated Fm), cucumber variety No. 6 (beijing academy of agricultural sciences) was selected, and the test method was the same as in example 3.
The disease classification standard of cucumber root rot is counted according to the following:
level 0: no symptoms;
stage 1: the main root turns slightly brown, has lateral root or stem base or yellow disease spot, and the plant grows normally;
3 stages: the main root is rotten, few lateral roots or stem bases have lesions, and the plant is slightly dwarfed and does not wilt;
5 stages: the main root is rotten, and no rot occurs to the basal part of the lateral root or stem; the plants wilt, grow weak, and obviously dwarf;
7 stages: the root system and the stem base are completely rotten, and the plant dies.
Incidence = (number of affected plants/total number of investigation) ×100%
Disease index=100×Σ (number of plants at each stage×relative number)/(total number of plants investigated×highest number of stages)
Relative control (%) = (control disease index-treatment disease index)/control disease index x 100%
By the method, obvious disease symptoms appear on cucumber seedlings after being inoculated with fusarium oxysporum and fusarium moniliforme, plants are obviously dwarfed, lesions with different degrees occur on roots, and disease indexes respectively reach 72.4 and 69.5. After the strain Y88 fermentation liquor is applied, the incidence and the disease index of cucumber root rot can be obviously reduced, the potted plant test results are shown in figures 11 and 12, the control effect of the strain Y88 on cucumber root rot on the central farm No. 6 can reach 48.9% and 26.7% respectively, and the control effect is not obviously different from that of the carbendazim treatment group which is 500 times 50%. However, for the cucumber root rot caused by different pathogenic bacteria, the influence of the strain Y88 and the carbendazim treatment group on the growth of cucumber plants is different, and for the cucumber root rot caused by fusarium oxysporum infection, the control effect of the strain Y88 and the carbendazim treatment group is higher, and the single plant fresh weight and the single plant dry weight of cucumber seedlings can be obviously improved. For cucumber root rot caused by fusarium moniliforme infection, the control effect of the strain Y88 and the carbendazim treatment group is slightly low, and the growth inhibition effect caused by diseases cannot be relieved. The results show that the antagonistic bacteria have obvious inhibition effect on cucumber root rot caused by fusarium oxysporum and fusarium moniliforme, and can be used as biocontrol actinomycetes for further research and application.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. Streptomyces salt houseStreptomyces sioyaensisY88, the preservation number is CGMCC NO. 27483.
2. A microbial inoculum comprising Streptomyces sioyaensis Y88 according to claim 1.
3. A biopesticide or biofertilizer prepared from the streptomyces salina Y88 or a microbial inoculum thereof according to claim 1.
4. A biocontrol or antibacterial agent prepared from the streptomyces salina Y88 of claim 1.
5. The use of the Streptomyces sioyaensis Y88 or a microbial inoculum thereof according to claim 1 for:
1) For antagonizing phytopathogenic fungi;
2) For antagonizing phytopathogenic bacteria;
3) Is used for preventing and treating wheat stem basal rot;
4) Is used for preventing and treating cucumber root rot;
5) Is used for preparing biological pesticides.
6. The use according to claim 5, wherein the phytopathogenic fungi comprise: fusarium pseudograminearumFusarium pseudograminearum) Fusarium oxysporum (F.oxysporum)Fusarium oxysporum) Fusarium moniliforme (F. Moniliforme)Fusarium moniliforme) Botrytis cinereaBotrytis cinerea) Rhizoctonia cerealis @Rhizoctonia cerealis) Rhinocerotis microorumCeratocystis fimbriata) Rhizoctonia solani of riceRhizoctonia solani) Tomato early blight germAlternaria solani) Lily root rot fungusFusarium oxysporum f.sp.lilii) Watermelon fusarium wiltFusarium oxysporum f.sp.niveum) Cotton fusarium wilt bacteriaVerticillium dahliae Kleb) Fusarium species of the round branchFusarium rotundiformis) Bacterial plaque of celerySeptoria apiicola Speg) The colletotrichum gloeosporioides isColletotrichum destructivum）；
The phytopathogenic bacteria include: cucumber angular leaf spot germPseudomonas syringae pv. lachrymans) Citrus canker pathogenXanthomonas citri) Black rot of chinese cabbageXanthomonas campestris pv.campestris) Chilli scab germXanthomonas campestris pv. vesicatoria) Bacterial wilt of eggplantRalstonia solanacearum）。
7. Use of the Streptomyces siosphaericus Y88 and/or bacteriostatic active substances produced thereof according to claim 1:
a) For combating pathogenic bacteria and related diseases or plant diseases caused by pathogenic bacteria;
b) For inducing plants to enhance resistance to related diseases or plant diseases caused by pathogenic bacteria;
c) Is used for preparing biocontrol agents, antibacterial agents or plant immunity inducing agents.
8. The use according to claim 7, wherein the pathogenic bacteria comprise plant pathogenic fungi, plant pathogenic bacteria;
the plant pathogenic fungi include: fusarium pseudograminearumFusarium pseudograminearum) Fusarium oxysporum (F.oxysporum)Fusarium oxysporum) Fusarium moniliforme (F. Moniliforme)Fusarium moniliforme) Botrytis cinereaBotrytis cinerea) Rhizoctonia cerealis @Rhizoctonia cerealis) Rhinocerotis microorumCeratocystis fimbriata) Rhizoctonia solani of riceRhizoctonia solani) Tomato early blight germAlternaria solani) Lily root rot fungusFusarium oxysporum f.sp. lilii) Watermelon fusarium wiltFusarium oxysporum f.sp.niveum) Cotton fusarium wilt bacteriaVerticillium dahliae Kleb) Fusarium species of the round branchFusarium rotundiformis) Bacterial plaque of celerySeptoria apiicola Speg) The colletotrichum gloeosporioides isColletotrichum destructivum）；
The phytopathogenic bacteria include: cucumber angular leaf spot germPseudomonas syringae pv. lachrymans) Citrus canker pathogenXanthomonas citri) Black rot of chinese cabbageXanthomonas campestris pv.campestris) Chilli scab germXanthomonas campestris pv. vesicatoria) Bacterial wilt of eggplantRalstonia solanacearum）。
9. The use according to claim 7, wherein the plant diseases comprise wheat stem rot, cucumber root rot.
10. The use according to any one of claims 5-9, wherein the plants comprise wheat, cucumber.
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