CN107904196B - Streptomyces yanshi and application thereof - Google Patents

Streptomyces yanshi and application thereof Download PDF

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CN107904196B
CN107904196B CN201711498986.XA CN201711498986A CN107904196B CN 107904196 B CN107904196 B CN 107904196B CN 201711498986 A CN201711498986 A CN 201711498986A CN 107904196 B CN107904196 B CN 107904196B
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谢江辉
起登凤
周登博
王飞
张妙宜
冯仁军
王尉
臧小平
井涛
陈宇丰
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Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
Haikou Experimental Station of Chinese Academy of Tropical Agricultural Sciences
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Abstract

The invention provides an actinomycete, which is Streptomyces villosus XJC-HDM11, and the preservation number is CCTCC NO: m2017493. The Streptomyces yanshi XJC-HDM11 has the growth pH range of 5-9, the growth temperature of 25-30 ℃, can not grow on a culture medium with the NaCl content of more than 3 percent, has antagonism on the race 1 and 4 of the banana fusarium wilt, particularly has remarkable antagonism on the banana fusarium wilt 4, has wide development space in the prevention and treatment of the banana fusarium wilt, has good development and application prospects, can effectively improve the chlorophyll content of bananas, promotes the growth and biomass accumulation of banana plants, and has good development and application prospects.

Description

Streptomyces yanshi and application thereof
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to streptomyces villosus and application thereof.
Background
Banana vascular wilt, also known as panama disease and banana xanthophyll disease, is a vascular bundle wilting disease caused by fusarium oxysporum cubense f.sp. Pathogenic bacteria invade from the wound of the root of the host, spread to upper leaves through the vascular bundle via the corms and the pseudostems, block the xylem catheter, bring obstacles to the water transportation of plants and cause the plants to wither and die. Once a land is attacked by the destructive soil-borne disease, the pathogenic bacteria spread quickly and are difficult to cure radically, so the disease is called banana cancer. Therefore, the prevention and treatment of banana vascular wilt is imminent.
At present, no effective prevention and treatment method exists for the disease, and no ideal chemical agent and high-quality disease-resistant variety are available. Some cultivation management measures can only play a local control role. Moreover, long-term use of some chemical bactericides and modifying agents can easily cause the problem of drug resistance of pathogenic bacteria to be prominent, destroy the ecological environment of soil and be unsafe for human beings. The microbial pesticide can utilize microbes to generate disease-resistant substances, compete with pathogenic bacteria for nutrition and space sites, induce plants to generate disease resistance and the like, safely and effectively prevent and treat plant diseases, and has important application value in prevention and treatment of the plant diseases.
In conclusion, the environment-friendly, efficient and safe microbial bactericide has a wide development space in the prevention and treatment of banana vascular wilt, and is also a requirement for agricultural safe and sustainable development.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides the actinomycete which is streptomyces villosus, has good antagonistic action on banana fusarium wilt, has wide development space in the prevention and treatment of the banana fusarium wilt, can effectively improve the chlorophyll content of bananas, promotes the growth and biomass accumulation of banana plants, and has good development and application prospects.
The first aspect of the invention provides an actinomycete, which is Streptomyces villosus XJC-HDM11, and the preservation number is CCTCC NO: m2017493.
In a second aspect of the present invention, there is provided a fermentation broth of actinomycetes or a filtrate of the fermentation broth or an ethanol extract of the fermentation broth as described in the first aspect of the present invention.
A third aspect of the present invention provides the use of an actinomycete according to the first aspect of the invention, or a fermentation broth of a strain according to the second aspect of the invention or a filtrate of the fermentation broth or an ethanol extract of the fermentation broth, for antagonizing Fusarium oxysporum f sp.
Further, the actinomycetes are used for antagonizing banana vascular wilt disease 1 and/or banana vascular wilt disease 4.
In a fourth aspect of the present invention, there is provided the use of the actinomycete according to the first aspect of the present invention, or the strain broth according to the second aspect of the present invention, or the filtrate of the broth, or the ethanol extract of the broth, for the control of banana vascular wilt.
A fifth aspect of the present invention provides the use of the actinomycete according to the first aspect of the invention, or a fermentation broth of the strain according to the second aspect of the invention, or a filtrate or ethanol extract of the fermentation broth, for increasing the chlorophyll content of banana.
In a sixth aspect of the invention there is provided the use of an actinomycete according to the first aspect of the invention, or a fermentation broth of a strain according to the second aspect of the invention, or a filtrate of the fermentation broth, or an ethanol extract of the fermentation broth, for promoting the growth of banana plants.
In a seventh aspect of the invention there is provided the use of an actinomycete according to the first aspect of the invention, or a fermentation broth of a strain according to the second aspect of the invention or a filtrate of a fermentation broth or an ethanol extract of a fermentation broth, for promoting biomass accumulation in bananas.
An eighth aspect of the present invention provides a microbial inoculum comprising the actinomycetes according to the first aspect of the present invention.
The Streptomyces yanshi XJC-HDM11(streptomyces yanii XJC-HDM11) has the growth pH range of 5-9, the growth temperature of 25-30 ℃, can not grow on a culture medium with the NaCl content of more than 3 percent, has antagonism on the 1 st and 4 th microspecies of the banana fusarium wilt, particularly has obvious antagonism on the 4 th banana fusarium wilt, has wide development space in the prevention and treatment of the banana fusarium wilt, has good development and application prospects, can effectively improve the chlorophyll content of bananas, promotes the growth and biomass accumulation of banana plants, and has good development and application prospects.
Drawings
FIG. 1 shows the morphology of spore hyphae (A) and spores (B) of strain XJC-HDM11
FIG. 2 is a phylogenetic tree of strain XJC-HDM11 and related strains constructed based on the 16S rDNA sequence.
FIG. 3 is the effect of Streptomyces yanshi XJC-HDM11 on the chlorophyll content of banana.
FIG. 4 is the effect of Streptomyces yandes XJC-HDM11 on the fresh weight of bananas.
FIG. 5 is a graph of the effect of 5 antagonistic actinomycetes XJC-HDM11 on banana dry weight.
Detailed Description
The invention will be better understood by reference to the following examples.
The invention provides an actinomycete, which is Streptomyces villosus XJC-HDM11(Streptomyces yaniiiXJC-HDM 11), and the preservation number is CCTCC NO: m2017493, the preservation date is 2017, 9 and 11 days, the preservation unit is China center for type culture Collection, and the address is Wuhan university in Wuhan, China. The Streptomyces yanshi XJC-HDM11 is separated from the rhizosphere soil of tea trees collected from Puer city in Yunnan province.
1 materials of the experiment
1.1 test soil
Collecting rhizosphere soil of tea trees in Puer city of Yunnan province, placing in a sterile sealing bag, mixing, sealing, and storing in an ice box. Collecting, removing impurities such as root system and stone, and storing in refrigerator at 4 deg.C for use.
1.2 test Medium
The test medium of the present experiment included a separation medium, a medium for observing culture characteristics, a physiological and biochemical medium, a fermentation medium, and the like (xuli, 2007; huangxiaolong, 2009).
TABLE 1 isolation Medium for Actinomycetes and its formulation
Figure BDA0001525992260000031
Figure BDA0001525992260000041
TABLE 2 culture characteristic observation culture medium and its formulation
Figure BDA0001525992260000042
Figure BDA0001525992260000051
TABLE 3 culture media for physiological and biochemical characteristics
Figure BDA0001525992260000052
Figure BDA0001525992260000061
TABLE 4 fermentation media and their formulations
Figure BDA0001525992260000062
Figure BDA0001525992260000071
1.3 reagents and instrumentation used in this experiment
(1) Primary reagent
TABLE 5 major Biochemical reagents and sources
Figure BDA0001525992260000072
(2) Apparatus and device
TABLE 6 Instrument and apparatus
Figure BDA0001525992260000073
Figure BDA0001525992260000081
1.4 test pathogens
The Fusarium oxysporum f.sp.Cubense (FOC 1) No. 1 and No. 4 banana Fusarium oxysporum (FOC 4) are used for screening anti-Fusarium active actinomycetes and determining the bacteriostatic activity.
2 method of experiment
2.1 isolation and identification of rhizosphere soil Actinomycetes
2.1.1 isolation and purification of rhizosphere soil Actinomycetes
Naturally air drying the soil sample, fully grinding and sieving, weighing 1g of the soil sample, dissolving in 10mL of sterile water, heating at 55 ℃ for 20min, and placing in a shaking table at 180r/min for culturing for 20min to prepare suspension. Taking supernatant, diluting by 10 times dilution method to obtain 10-1、10-2And 10-3In the soil suspensionRespectively sucking 0.1mL of suspension, coating the suspension on a separation culture medium, performing inverted culture at 28 ℃ for 2-4 weeks, setting 3 times of repetition for each gradient, and selecting different single colonies and performing repeated purification on a YE culture medium by a streaking method.
2.1.2 screening of antagonistic bacteria
Plate confrontation culture (grandson waves, 2010): using a puncher with the diameter of 5mm to take the fungus cakes inoculated with 5d and consistent growth force of FOC4 pathogenic bacteria edges, inoculating the fungus cakes to the center of each PDA plate, inoculating the bacteria to be tested at a position 2.5cm away from the center of the colony of the pathogenic bacteria, inoculating 4 test strains to each dish, taking the pathogenic bacteria inoculated with FOC4 only as a control, placing the inoculated pathogenic bacteria in an incubator at 28 ℃ for inverted culture for 7d, and observing the result.
2.1.3 determination of in-dish antibiogram
The bacterial strain is subjected to bacteriostasis spectrum determination by adopting a confrontation culture method (plum blossom King, 1997): a5 mm puncher is used for taking a bacterial cake of 2 purified plant pathogenic bacteria, the bacterial cake is inoculated to the center of a PDA (personal digital assistant) flat plate, a small amount of bacteria to be detected are respectively inoculated to four points 2.5cm away from the bacterial cake of the pathogenic bacteria, a culture dish only inoculated with the pathogenic bacteria is used as a blank control group, and each treatment is repeated for 3 times. After culturing in an incubator for 4-7 days, measuring the colony growth diameter of pathogenic bacteria to be tested by adopting a cross measurement method, and counting the bacteriostasis rate (Xiexing, 2011; summer dragon fungus, 2013) according to the following formula:
colony diameter (mm) — average value of colony diameters measured-5.0
Figure BDA0001525992260000091
2.1.4 culture characteristic Observation of antagonistic Strain
Observations of the culture characteristics were made by reference to the standard medium used in the international Streptomyces program for the culture characteristics of Actinomycetes (Cui BS, 2008). Inoculating antagonistic actinomycetes on ISP2, ISP3, ISP4, ISP5, ISP6 and ISP7 culture media, culturing at 28 ℃ for 7-21d, and observing and recording culture characteristics of the strains on each culture media, including characteristics of colony morphology, aerial hypha production, spore color and intrastromal hypha color.
2.1.5 scanning Electron microscopy
Soaking the cover glass with potassium dichromate with the concentration of 0.05g/L, soaking and eluting with alcohol, washing with ultrapure water, blow-drying, and sterilizing at 121 ℃ for 20 minutes. The sterilized coverslip was inserted at 45 ℃ onto the culture medium of Goodpasture No. I inoculated with actinomycete strain, and cultured at 28 ℃ for 7-10 days. And (4) conveying the cover glass sample to a detection center for observation of a scanning electron microscope, placing the cover glass sample with the bacteria in a vacuum coating machine for spraying gold coating, and observing the fine structures on the surfaces of hyphae and spores of the strains by using the scanning electron microscope.
2.1.6 measurement of physiological and biochemical Properties
The physiological and biochemical identification of the strain was carried out by referring to the methods of Shirking (1966) and Xuelihua et al (2007) mainly in the following respects.
(1) Determination of enzymatic Properties
① urease test:
inoculating the strain on a urease culture medium, culturing for 4d at 28 ℃, and observing whether the culture medium is discolored. The test strains were tested for their ability to produce urease, and the medium was positive for pink and negative for no white.
② esterase (Tween 20, Tween 80) assay:
streaked onto esterase medium for 1-2 weeks with daily observation. Positive if there is a faint halo around its growth, and negative if there is no halo.
③ hydrolysis of starch:
nutrient agar is used as a basic culture medium, and 1.0% of soluble starch is added. Inoculating the test strain on a flat plate, adopting a point inoculation method (the inoculation diameter is not more than 5mm), and when the strain grows well, dripping iodine solution around the bacterial colony for detection. If a transparent circle is generated around the strain, the generation of amylase is indicated, and the size of the circle indicates the strength of the amylase activity; if no amylase is produced, the color is blue.
④ liquefaction of gelatin:
the strain was inoculated on the surface of gelatin medium without puncturing the medium, cultured at 28 ℃ and observed for the degree of liquefaction of the medium at 5d, 10d, 20d and 30d, respectively. Before observation, the test tube needs to be cooled for 20-30min or washed by tap water for 30min, and the liquefaction degree of the culture medium can be observed.
⑤ cellulose decomposition:
immersing one end of the filter paper strip in a liquid culture medium, inoculating the strain to be detected on the filter paper sheet above the liquid level after sterilization, and observing whether the filter paper strip is decomposed or not after one month.
⑥ nitrate reduction:
the bacterial strain to be tested is inoculated in a nitrate reduction culture medium and cultured for 7 and 14 days at the temperature of 28 ℃, and the culture medium without inoculation is used as a control. A small amount of culture medium cultured for 7d and 14d was added to the test tube, and one drop of solution A and solution B was added, as was the case with the control. When the solution becomes pink, rose red, orange or brown, etc., the nitrate is positive in reduction; if no red color appears, 1 or 2 drops of diphenylamine reagent is dripped, and if the reagent is blue, the reduction effect is negative; if not blue, it is still treated as positive.
(2) Experiment for Using Single carbon Source
The carbon sources selected in the test are D-fructose, xylose, rhamnose, arabinose, raffinose, melezitose, anhydrous lactose, D-galactose, α -lactose, D-trehalose, D-mannose, D-ribose, inositol, sorbitol, mannitol, salicin and soluble starch, the carbon sources are added into a basic culture medium of Pugodi according to the concentration of 1% of the carbon sources, the strains to be tested are inoculated, the strains to be tested are cultured at constant temperature of 28 ℃ for 7-14D, the strains inoculated by the basic culture medium without any carbon source are used as blank control, the growth condition of the strains is observed, if the strains can grow, the strains can utilize the carbon sources, and if the strains cannot grow, the strains can not utilize the carbon sources.
(3) Experiment for utilization of Single Nitrogen Source
Nitrogen sources selected for the test: histidine, methionine, serine, oxamic acid, glycine, hydroxyproline, phenylalanine, glutamic acid, cysteine, arginine, valine, ammonium molybdate tetrahydrate, ammonium acetate, ammonium nitrate, ammonium sulfate were added to the basal medium at a concentration of 0.5%. Inoculating strains, culturing at constant temperature of 28 deg.C for 7-14d, taking strains inoculated by basal medium without any nitrogen source as blank control, and observing growth condition of the strains. If the strain can grow, the strain can utilize the nitrogen source; if the strain can not grow, the strain can not utilize the nitrogen source.
(4) Measurement of other physiological and biochemical parameters
① temperature tolerance test:
inoculating the strain to be tested in the same culture medium, culturing at 20 deg.C, 24 deg.C, 28 deg.C, 32 deg.C and 36 deg.C for 7-14d under the condition of identical culture conditions, observing and recording the growth condition of bacterial colony, thereby determining the optimum temperature for strain growth.
② pH tolerance test:
the strains to be tested were inoculated into liquid media with pH values of 4, 5, 6, 7, 8, 9, and 10, respectively, and the other culture conditions were kept consistent, and the culture was carried out at 28 ℃ with observation every other week for four weeks. The growth of the strain was observed and recorded each time to determine the optimum pH for growth of the strain.
③ salt tolerance test:
respectively inoculating the strains to be detected on NaCl (1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%) culture media with different concentrations, wherein other nutrient components of the culture media are the same, culturing at 28 ℃, taking 7 days as an observation period, observing for 4 weeks, and recording whether the strains can grow so as to determine the upper and lower limit concentrations of NaCl which can be tolerated by the strains.
④ experiment for production of hydrogen sulfide:
inoculating a strain to be detected on a hydrogen sulfide culture medium containing ammonium ions in a certain proportion, and culturing for 2 weeks at 28 ℃, wherein if the culture medium is black, hydrogen sulfide is generated; and if no black color exists, no hydrogen sulfide is generated. 2.1.7 molecular biological identification of antagonistic strains
(1) Extraction of actinomycete genomic DNA
Total DNA was extracted using biotek's kit for rapid extraction of bacterial genomic DNA (DP1301, Beijing Baitach Biotech Co., Ltd., China).
(2) Sequencing and analysis of 16S rDNA
① 16 PCR amplification of S rDNA:
PCR amplification was performed using actinomycete genomic DNA as a template and universal primers 27F and 1492R. The primer sequences are as follows: the upstream primer 27F (5 'AGAG TTTG ATCC TGGC TCAG 3'), and the downstream primer 1492R (5 'TACG GCTACCTTGTTACGAC TT 3'). The specific reaction system is shown in Table 7, and the reaction procedures (Zhoujun nu, 2014; Heufaphenanthrene, 2015; Na Yua, 2014) are shown in Table 8.
PCR reaction system of table 716S rDNA gene
Figure BDA0001525992260000121
TABLE 816 PCR amplification reaction conditions for S rDNA genes
Figure BDA0001525992260000122
② electrophoretic detection of PCR products:
after the PCR reaction is finished, 5 mu L of PCR amplification product is taken to carry out electrophoresis detection on the PCR product of the strain on 1% agarose gel, and whether the connection is successful is determined according to the length of the target fragment.
③ sequencing and construction of phylogenetic trees:
the PCR product of the strain was sent to Huada Gene Co for sequencing. The determined gene sequences were aligned using BLAST software and compared for homology to the known 16S rDNA in GenBank and ezbiocoud databases. Sequences with higher homology are found out for multiple matching array analysis, and MEGA5.1 software is adopted to carry out clustering analysis and phylogenetic tree construction by a Neighbor-Joining method (Na Yua, 2014; Jianghua, 2015).
2.2 evaluation of bacteriostatic Activity of antagonistic Strain
2.2.1 determination of plate-confronted broad-spectrum antibacterial activity
Performing broad-spectrum determination on the strains by adopting a plate confrontation method: and (3) taking a purified fungus cake of 2 plant pathogenic bacteria by using a 5mm puncher, inoculating the fungus cake to the center of a PDA (personal digital assistant) flat plate, respectively inoculating a small amount of bacteria to be detected at four points 2.5cm away from the pathogen fungus cake, and repeating the treatment 3 times by using a culture dish only inoculated with the pathogenic bacteria as a blank control group. After culturing in an incubator for 4-7 days, measuring the colony growth diameter of pathogenic bacteria to be tested by adopting a cross method, and counting the bacteriostasis rate (Xiexing, 2011; Xiongdan, 2013) according to the following formula:
colony diameter (mm) — average value of colony diameters measured-5.0
Figure BDA0001525992260000131
2.2.2 measurement of bacteriostatic Activity of Secondary metabolites against pathogenic bacteria
Respectively inoculating the strains into an SLM liquid culture medium for culturing for 7 days, adding absolute ethyl alcohol, oscillating for 10 days, carrying out vacuum filtration on 5 layers of filter paper, and collecting secondary metabolites of the strains as samples. A bacteriostatic paper disc method (xu Tertiary cloud, etc. 2003; Wellman-Labadie et al,2010) is adopted, a sterilized culture medium is poured into a flat plate, after the flat plate is solidified, a filter paper disc with the diameter of 9mm is prepared by a puncher, the filter paper disc is subjected to dry heat sterilization for 2h, the filter paper disc is soaked in a sample in an aseptic operation table for 30min, the solvent is taken out and volatilized, the filter paper disc is flatly pasted on the medicine-containing flat plate, and meanwhile, indicator bacteria are inoculated in the center of the culture medium. And finally, placing all culture dishes in a constant-temperature incubator at 28 ℃ for culture, observing every day, recording and photographing, and calculating the average value of the diameters of the inhibition zones by making three parallels for each group.
2.3 preparation of Strain fermentation broth
Preparing fermentation liquor: the nitrogen content of the soybean meal is 7.0 percent, the C/N is 6.76 percent and the carbon content of the molasses is 20.27 percent according to the measurement of agricultural academy of sciences in Haikou city. In the test, according to the carbon-nitrogen ratio of the soybean meal and the carbon content of the molasses, the carbon-nitrogen ratio of a matrix is adjusted to be 25:1, the total amount of the molasses and the soybean meal is 250g, namely 34.25g of the soybean meal, 215.75g of the molasses and 1000mL of sterile water, a molasses and cake fertilizer mixed solution is prepared, and the mixed solution is filled into a 5L triangular flask for testing.
And (3) flora construction: preparing YE liquid culture medium, subpackaging in 250mL triangular flasks, sterilizing at 121 ℃ for 20 minutes, cooling, inoculating fresh test strains, performing shake culture at 28-30 ℃ at 180rpm/min for 3 days, inoculating 5% of strain inoculation amount into newly-prepared fermentation nutrient solution, and performing fermentation culture at 28-30 ℃ for 9d under 180rpm/min shake culture for testing.
2.4 potted plant test of antagonistic Actinomycetes against Banana wilt
The pot experiment is carried out in a greenhouse of a research institute of tropical biotechnology of the Chinese tropical agricultural academy of sciences in 2017 in 5-7 months, and the greenhouse conditions are controlled as follows: the temperature is about 28 ℃, the humidity is 70 percent, and the natural illumination is carried out. The experiment was set to 4 treatments, CK 1: applying sterile water without inoculating pathogenic bacteria; CK 2: inoculating pathogenic bacteria, and applying sterile water; CK 3: inoculating pathogenic bacteria, namely, prednisone; a: inoculating pathogenic bacteria, and applying fermentation liquor without adding bacteria; AB: inoculating pathogenic bacteria, and applying strain XJC-HDM11 fermentation liquor.
Inoculating by root-damaging and bacterium-immersing method. Selecting banana seedlings with consistent growth vigor and 5-6 leaves, cutting off a second main root at a concentration of 105Soaking in the pathogenic bacteria spore suspension per mL for 30min, transplanting into a plastic pot with the soil loading of 700g, and irrigating 20mL of the pathogenic bacteria suspension at the rhizosphere soil of the banana seedlings, wherein each treatment is repeated for 3 times, and each time, 20 banana seedlings are repeated.
And (5) counting disease indexes. And (4) observing the disease condition after inoculating pathogenic bacteria by adopting a fractional counting method, and counting the disease index by starting to count the infection of the first banana seedling. After the banana seedlings are completely infected with diseases, applying fermentation liquor diluted by 100 times according to different treatments, wherein 200mL of each banana seedling is treated, and applying the same amount of clear water to CK; the fermentation liquor and the clear water are repeatedly applied every 15 days for 3 times in total.
During the test period, other management measures of each treatment are consistent. And (5) recording the number of etiolated leaves and normal leaves of each treated banana at the 60 th day of banana transplantation, and calculating disease index and prevention and control effect. The grade standard of banana wilt is 0 grade, and plants are healthy; grade 1, with 25% yellowing leaves; grade 3, 25 to 50 percent of etiolation diseased leaves; grade 5, 50-90% of etiolated diseased leaves; and 7, the leaves are all yellow, and the plants die. Calculating the disease index and the disease prevention effect of the bananas:
Figure BDA0001525992260000141
Figure BDA0001525992260000142
2.5 investigation and determination of physiological indexes of banana plants
Respectively transplanting the banana seedlings for 0d, 15d, 30d, 45d and 60d, and measuring the chlorophyll content of the second fully-unfolded leaf on the uppermost part of each treatment by adopting an ethanol extraction method; at 60d, the leaf area of the single plant, the length and diameter of the root system, the plant height, the false stem circumference, the biomass of the overground part and the biomass of the root system are measured.
3 results and analysis
3.1 screening and morphological characterization of Actinomycetes
Carrying out plate coating separation and streaking on the purified strains, removing the weight according to the colony morphology and the color of the strains on a purification culture medium, and carrying out primary screening by a plate opposition culture method and secondary screening by an Oxford cup method to obtain 1 actinomycete generating the largest inhibition zone; numbered XJC-HDM 11.
TABLE 9 culture characteristics of Strain XJC-HDM11 on 6 media
Figure BDA0001525992260000151
3.2 physiological and Biochemical characteristics of the Strain
TABLE 10 part physiological-biochemical characteristics of Strain XJC-HDM11
Figure BDA0001525992260000152
Figure BDA0001525992260000161
Positive result; negative result.
3.3 phylogenetic characteristics of the strains
Extracting total DNA of the strain XJC-HDM11, obtaining a 16S rDNA sequence of about 1.5kb through PCR amplification, sequencing to obtain a sequence thereof, submitting sequence information to EzTaxon for gene sequence similarity search to obtain sequence information of 9 strain model bacteria which have highest homology with the strain XJC-HDM11 and are named totally, performing phylogenetic analysis, and constructing a phylogenetic tree (figure 2). As can be seen from the figure, XJC-HDM11 and Streptomyces form a branch, and the homology of the 16S rDNA sequence is between 98.95 percent and 99.56 percent; among them, the homology with Streptomyces yanii was the highest and was 99.42%.
Combining morphological characteristics, physiological and biochemical characteristics and the analysis result of 16s DNA molecular sequence, the strain XJC-HDM11 is preliminarily identified as the Streptomyces yanii (Streptomyces yanii).
3.4 evaluation of the antibacterial Activity of Strain XJC-HDM11
3.4.1 antagonistic action of the plates on pathogenic bacteria
TABLE 11 inhibitory Effect of Strain XJC-HDM11 on pathogenic fungi
Figure BDA0001525992260000162
The data in the table are mean ± standard deviation. The different small letters in the same column indicate significant differences at P < 0.05 as tested by Duncan's New Complex Pole Difference method.
3.4.2 bacteriostatic activity of crude extract of strain XJC-HDM11 on hypha growth of pathogenic bacteria
TABLE 12 inhibitory Effect of crude extracts of Strain XJC-HDM11 on pathogenic fungi
Figure BDA0001525992260000171
3.4.3 prevention and control effects of the XJC-HDM11 fermentation liquid on banana vascular wilt
The incidence of each treated banana plant in the pot test is shown in table 12. The results show that the difference between the disease index and the prevention and control effect of different treatment of banana seedling blight is not obvious. The banana seedlings do not have disease symptoms and grow well by applying sterile water (CK1) without inoculating pathogenic bacteria. The disease indexes of inoculated pathogenic bacteria, applied clear water (CK2) and applied fermentation liquor (A) without added bacteria are both very high, which are 84.38 and 70.45 respectively; the disease index of the inoculated antagonistic bacteria XJC-HDM11 fermentation liquor (AB) is 10.23, and the prevention and control effect is as high as 83.12%.
TABLE 13 potted plant control effect of antagonistic actinomycetes XJC-HDM11 on banana fusarium wilt
Figure BDA0001525992260000172
The data in the table are mean ± standard deviation different small letters after the same column of data indicate significant differences at P < 0.05 as tested by Duncan's new double polar difference method.
3.4.4 Effect of the fermentation broth of Strain XJC-HDM11 on the chlorophyll content of Banana leaves
As shown in fig. 3, for 5 treatments, the content of chlorophyll of banana increased with the increase of transplanting time, the content of chlorophyll of CK1 increased continuously, the content of chlorophyll of CK2 decreased, both CK3 and A, AB treatments decreased first and then increased, while the lowest critical point of the decrease of AB treatment was higher than that of CK3 and a treatment, and the increasing trend was also significantly higher than that of the other 3 treatments. Because the chlorophyll content is in negative correlation with the morbidity, the CK1 treatment prevention and control effect is the worst, 20 banana seedlings are all infected with diseases, the disease index is continuously increased along with the increase of time, and the chlorophyll content in the treated banana seedlings is also reduced; the CK1 treats unanswered pathogenic bacteria, the banana seedlings grow healthily after being transplanted, but the chlorophyll content only shows a slow growth trend, and the maximum content is 0.99 mg/g; the CK3 and A are treated, although chlorophyll is in a growth trend in the later period, due to the infection, the growth point is always lower than that of a blank control CK1 which is not infected with diseases, and the growth point is respectively 0.81mg/g and 0.68 mg/g; on the contrary, the AB treatment has the antibacterial effect, the disease index of the banana seedlings at the later stage is reduced, the disease prevention effect is obvious, chlorophyll is in a straight line rapid growth trend, and the concentration of chlorophyll is as high as 1.28mg/g at 60 days, which is obviously higher than that of other treatments. This indicates that the fermentation culture of the antagonistic bacteria XJC-HDM11 is beneficial to improving the chlorophyll content of the bananas.
2.4.5 Effect of Strain XJC-HDM11 fermentation broth on Banana plant growth
As can be seen from Table 14, the leaf area of the banana seedlings treated with fenaminosulf (CK3) (1)155.42cm2) The root length (1036.92cm), the root diameter (0.78mm), the plant height (46.22cm) and the stem thickness (4.97cm) are obviously higher than those of the other 3 treatments, the physiological indexes of the A treatment are only higher than those of CK2 treatment which is completely infected, and are obviously lower than those of AB treatment, CK3 treatment and CK1 treatment, the banana seedlings treated by CK2 have various obvious physiological indexes, are short and small, and the leaves are yellow and small. While the leaf area (1398.13 cm) of the banana seedlings treated by the fermentation liquor of the antagonistic strain XJC-HDM112) The root length (1354.87cm), the root diameter (1.11mm), the plant height (53.76cm) and the stem thickness (6.19cm) are all obviously higher than other treatments, which shows that the antagonistic bacteria XJC-HDM11 fermentation liquid not only has disease resistance activity, but also has the growth promotion effect on banana seedlings, can promote the leaf area of plants, enhance photosynthesis, increase the growth of root systems and promote transpiration, thereby promoting the growth of banana plants and increasing the yield.
TABLE 14 growth Effect of antagonistic actinomycetes XJC-HDM11 on Banana plants
Figure BDA0001525992260000181
The data in the table are mean ± standard deviation. Different small letters after the same column of data indicate significant differences at P < 0.05 levels as tested by Duncan's New Complex Pole Difference.
2.4.6 Effect of Strain XJC-HDM11 fermentation broth on Banana plant Biomass
As can be seen from FIGS. 4 and 5, the fresh weight and the dry weight of the aerial parts of the 5 treated banana seedlings are AB & gt CK1 & gt CK3 & gt A & gt CK2, the AB treatment is significantly higher than the other 4 treatments, the fresh weight of the aerial parts is 48.20g, the dry weight is 4.34g, and the water content is 90.99%. The fresh weight of underground parts, AB treatment and CK1 have no significant difference and are significantly higher than other 3 treatments, and CK3 is significantly higher than CK1 and CK2, which shows that CK3 treatment has certain control effect but is inferior to AB treatment; the dry weight of the underground part, AB treatment was significantly higher than the other treatments, with no significant difference between CK1 treatment and CK3 treatment, and also significantly higher than CK2 and a treatments. After the AB fermentation broth is treated, the dry matter accumulation is also obviously improved and is higher than that of normal growing banana seedlings, which shows that XJC-HDM11 fermentation broth has promotion effect on banana growth and biomass accumulation, and the effect is obvious.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims (9)

1. The actinomycete is Streptomyces villosus XJC-HDM11, and the preservation number is CCTCC NO: m2017493.
2. A fermentation broth of the actinomycete of claim 1, or a filtrate of the fermentation broth, or an ethanol extract of the fermentation broth.
3. Use of the actinomycete according to claim 1, or a fermentation broth or a filtrate of a fermentation broth or an ethanol extract of a fermentation broth of the actinomycete according to claim 2 for antagonizing fusarium oxysporum f.sp.
4. Use according to claim 3, wherein the actinomycete is used to antagonize Banana wilt bacteria No. 1, and/or Banana wilt bacteria No. 4.
5. Use of the actinomycete according to claim 1, or a fermentation broth or a filtrate of a fermentation broth or an ethanol extract of a fermentation broth of the actinomycete according to claim 2 for controlling banana vascular wilt.
6. Use of the actinomycete according to claim 1, or a fermentation broth or a filtrate of a fermentation broth or an ethanol extract of a fermentation broth of the actinomycete according to claim 2 to increase the chlorophyll content of bananas.
7. Use of the actinomycete according to claim 1, or a fermentation broth or a filtrate of a fermentation broth or an ethanol extract of a fermentation broth of the actinomycete according to claim 2 for promoting the growth of banana plants.
8. Use of the actinomycete according to claim 1, or a fermentation broth or a filtrate of a fermentation broth or an ethanol extract of a fermentation broth of the actinomycete according to claim 2 to promote banana biomass accumulation.
9. A microbial preparation comprising the actinomycetes according to claim 1.
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