CN107904196A - A kind of Yan Shi streptomycetes and its application - Google Patents

Abstract

The present invention provides an actinomycete, which is Streptomyces yanii XJC-HDM11, and the preservation number is CCTCC NO: M 2017493. Streptomyces yanii XJC-HDM11 of the present invention has a growth pH range of 5-9, a growth temperature of 25-30° C., and cannot grow on a medium with a NaCl content greater than 3%, and is effective against No. 1 and No. 4 races of Fusarium wilt of banana All have antagonistic effects, especially the antagonistic effect on Fusarium wilt of banana No. 4 is significant. It has broad development space in the prevention and treatment of Fusarium wilt of banana, has good development and application prospects, and can effectively increase the chlorophyll content of bananas and promote the growth of banana plants. growth and biomass accumulation, and has good development and application prospects.

Description

A kind of Streptomyces yanii and its application

technical field

The invention belongs to the field of microorganisms, and in particular relates to a Streptomyces yanii and its application.

Background technique

Banana Fusarium wilt, also known as Panama disease and banana yellow leaf disease, is a vascular wilting disease caused by Fusarium oxysporum f.sp.cubense. The pathogen invades from the root wound of the host, and spreads to the upper leaves through the corm and pseudostem through the vascular bundle. The pathogen blocks the xylem vessel, hinders the water transportation of the plant, and causes the plant to wither and die. This devastating soil-borne disease, once a field develops, the germs spread quickly and are difficult to cure, so it is called "banana cancer". Therefore, it is imminent to control banana wilt.

At present, there is no effective control method for the disease, and there are no ideal chemical agents and high-quality disease-resistant varieties. Some cultivation management measures can only play a partial control role. And long-term use of some chemical fungicides and modifiers can easily lead to the problem of drug resistance of pathogenic bacteria, and damage the ecological environment of the soil, which is not safe for humans. Microbial-derived pesticides can use microorganisms to produce disease-resistant substances, compete with pathogenic bacteria for nutrients and space sites, and induce plant disease resistance to safely and effectively prevent and control plant diseases, which has important application value in the prevention and control of plant diseases.

To sum up, this environmentally friendly, efficient and safe microbial fungicide has broad development space in the control of banana wilt, and is also the need for safe and sustainable development of agriculture.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art, to provide a kind of actinomycetes, it is a kind of Streptomyces yanii, has good antagonism to banana wilt pathogen, has broad development in the control of banana fusarium wilt In addition, the actinomycetes can effectively increase the chlorophyll content of bananas, promote the growth and biomass accumulation of banana plants, and have good development and application prospects.

The first aspect of the present invention is to provide an actinomycete, which is Streptomyces yanii XJC-HDM11, and the preservation number is CCTCC NO: M 2017493.

The second aspect of the present invention is to provide the fermented liquid of actinomycetes as described in the first aspect of the present invention or the filtered liquid of the fermented liquid or the ethanol extract of the fermented liquid.

A third aspect of the present invention is to provide actinomycetes as described in the first aspect of the present invention, or the strain fermentation liquid or fermentation liquid or the filtrate of the ethanol extract of fermentation liquid as described in the second aspect of the present invention. Application in antagonizing Fusarium wilt of banana.

Further, the actinomycete is applied to antagonize Banana Fusarium wilt No. 1 and/or Banana Fusarium wilt No. 4.

The fourth aspect of the present invention is to provide actinomycetes as described in the first aspect of the present invention, or the bacterial strain fermented liquid described in the second aspect of the present invention or the filtrate of fermented liquid or the ethanol extract of fermented liquid in Application in the control of banana fusarium wilt.

The fifth aspect of the present invention is to provide actinomycetes as described in the first aspect of the present invention, or the bacterial strain fermented liquid described in the second aspect of the present invention or the filtrate of fermented liquid or the ethanol extract of fermented liquid in Application to increase the chlorophyll content of bananas.

The sixth aspect of the present invention is to provide actinomycetes as described in the first aspect of the present invention, or the bacterial strain fermented liquid described in the second aspect of the present invention or the filtrate of fermented liquid or the ethanol extract of fermented liquid in Application in promoting the growth of banana plants.

The seventh aspect of the present invention is to provide actinomycetes as described in the first aspect of the present invention, or the bacterial strain fermentation liquid or the filtrate of the fermentation liquid or the ethanol extract of the fermentation liquid as described in the second aspect of the present invention. Application in promoting banana biomass accumulation.

The eighth aspect of the present invention is to provide a bacterial agent, which contains the actinomycetes described in the first aspect of the present invention.

Streptomyces yanii XJC-HDM11 (streptomyces yanii XJC-HDM11) growth pH range of the present invention is 5-9, and growth temperature is 25-30 ℃, can not grow on the medium that NaCl content is greater than 3%, to banana fusarium wilt Both No. 1 and No. 4 races have antagonistic effects, especially No. 4, which has a significant antagonistic effect on banana Fusarium wilt. It has broad development space and good development and application prospects in the prevention and treatment of banana Fusarium wilt, and can effectively improve the quality of bananas. The content of chlorophyll can promote the growth and biomass accumulation of banana plants, and has a good development and application prospect.

Description of drawings

Fig. 1 is the morphology of sporocystae (A) and spores (B) of strain XJC-HDM11

Figure 2 is a phylogenetic tree of strain XJC-HDM11 and related strains constructed based on 16S rDNA sequence.

Figure 3 shows the effect of Streptomyces yanii XJC-HDM11 on the chlorophyll content of bananas.

Figure 4 shows the effect of Streptomyces yanii XJC-HDM11 on the fresh weight of bananas.

Figure 5 shows the effect of 5 antagonistic actinomycetes XJC-HDM11 on the dry weight of bananas.

Detailed ways

The present invention will be further described below in conjunction with specific examples, so as to better understand the present invention.

The invention provides an actinomycete, which is Streptomyces yanii XJC-HDM11 (streptomyces yaniiXJC-HDM11), the preservation number is CCTCC NO: M 2017493, the preservation date is September 11, 2017, and the preservation unit is China Typical Culture Collection Center at Wuhan University, Wuhan, China. The Streptomyces yanii XJC-HDM11 of the present invention is isolated from tea tree rhizosphere soil collected from Pu'er City, Yunnan Province.

1 Experimental materials

1.1 Test soil

The rhizosphere soil of tea trees in Pu'er City, Yunnan Province was collected, mixed evenly in a sterile sealed bag, sealed, and stored in an ice box. After collection, remove roots, stones and other sundries, and store them in the refrigerator at 4°C for later use.

1.2 Test medium

The test medium used in this experiment includes isolation medium, culture medium for observation of culture characteristics, physiological and biochemical medium and fermentation medium (Xu Lihua, 2007; Huang Xiaolong, 2009).

Table 1 The isolation medium and formula of actinomycetes

Table 2 culture characteristics observation medium and its formula

Table 3 Medium required for observation of physiological and biochemical characteristics

Table 4 fermentation medium and its formula

1.3 Reagents and equipment used in this experiment

(1) Main reagents

Table 5 Main biochemical reagents and their sources

(2) Instruments and equipment

Table 6 Instruments and Equipment

1.4 Pathogens tested

Fusarium oxysporum f.sp. Cubense, FOC1 and Fusarium oxysporum f.sp. Cubense, FOC4, for the activity of actinomycetes against Fusarium oxysporum Screening and assay of antibacterial activity.

2 Experimental methods

2.1 Isolation and identification of actinomycetes in rhizosphere soil

2.1.1 Isolation and purification of rhizosphere soil actinomycetes

Naturally air-dry the soil sample, fully grind it and sieve it, weigh 1g of the soil sample and dissolve it in 10mL of sterile water, heat it at 55°C for 20min, and place it on a shaker at 180r/min for 20min to make a suspension. Take the supernatant and dilute it by 10-fold dilution method, prepare soil suspensions of 10 ^-1 , 10 ^-2 and 10 ^-3 , absorb 0.1mL of the suspension and spread it on the separation medium, and invert at 28°C Cultivate for 2-4 weeks, set 3 replicates for each gradient, pick different single colonies and use the streak method for repeated purification on YE medium.

2.1.2 Screening of antagonistic bacteria

Adopt plate confrontation culture method (Sun Jianbo, 2010): Use a puncher with a diameter of 5mm to take the bacteria cake at the edge of the FOC4 pathogen that has been inoculated for 5 days and grow uniformly, and place it in the center of each PDA plate. Inoculate the bacteria to be tested at 2.5cm in the center, inoculate 4 test strains in each plate, take the bacteria inoculated only with FOC4 as a control, place them in an incubator at 28°C and incubate them upside down for 7 days, and observe the results.

2.1.3 Determination of antimicrobial spectrum in the dish

Adopt confrontation culture method (Li Shuzheng, 1997) to carry out antibacterial spectrum measurement to bacterial strain: Utilize 5mm puncher to take the bacterium cake of the pathogenic bacterium of 2 kinds of plants that have purified, and inoculate in the center of PDA flat board, respectively in the distance of pathogenic bacterium A small amount of bacteria to be tested was inoculated on four spots at 2.5 cm of the cake, and the petri dish only inoculated with pathogenic bacteria was used as the blank control group, and each treatment was repeated 3 times. After culturing in the incubator for 4-7 days, the colony growth diameter of the pathogenic bacteria tested was measured by the cross measurement method, and the antibacterial rate was calculated according to the following formula (Xie Ying, 2011; Xia Longsun, 2013):

Colony diameter (mm) = average value of measured colony diameter - 5.0

2.1.4 Observation of culture characteristics of antagonistic strains

Observation of culture characteristics was carried out by referring to the standard medium used in the description of the culture characteristics of actinomycetes in the International Streptomyces Program (Cui BS, 2008). Antagonistic actinomycetes were inoculated on ISP2, ISP3, ISP4, ISP5, ISP6, ISP7 medium, cultured at 28°C for 7-21 days, and the culture characteristics of the strains on each medium were observed and recorded, including colony morphology, The characteristics of the production of aerial hyphae, the color of spores, and the color of hyphae in the substrate.

2.1.5 SEM observation

Soak the coverslip in 0.05g/L potassium dichromate, soak and elute with alcohol, rinse with ultrapure water, blow dry, and sterilize at 121°C for 20 minutes. Insert the sterilized coverslip at 45° on the Gaoshi No. 1 medium connected with actinomycete strains, and culture at 28°C for 7-10 days. Sent to the inspection center for scanning electron microscope observation, put the cover glass sample with bacteria in the vacuum coating machine for gold spray coating, and use the scanning electron microscope to observe the fine structure of the mycelium and spore surface of the strain.

2.1.6 Determination of physiological and biochemical characteristics

Refer to the method of Shirking (1966) and Xu Lihua et al. (2007) to carry out physiological and biochemical identification of the strain, mainly in the following aspects.

(1) Determination of enzymatic properties

①Urease test:

The strain was inoculated on the urease medium, and after culturing at 28°C for 4 days, it was observed whether the medium changed color. Test the ability of the tested strain to produce urease. If the culture medium becomes pink, it is positive, and if it is not white, it is negative.

②Esterase (Tween 20, Tween 80) experiment:

Streak inoculation on esterase medium, culture for 1-2 weeks, and observe every day. Positive if there is a faint halo around its growth, negative if there is no halo.

③Starch hydrolysis:

Nutrient agar was used as the basic medium, and 1.0% soluble starch was added. Inoculate the strains to be tested on a flat plate, and adopt the spot connection method (the diameter of the inoculation should not exceed 5mm). When the strains grow well, add iodine solution around the colonies for detection. If there is a transparent circle around the strain, it means that amylase is produced, and the size of the circle indicates the strength of amylase activity; if no amylase is produced, it is blue.

④ Gelatin liquefaction:

The strains were inoculated on the surface of the gelatin medium without piercing the medium, cultured at 28°C, and the liquefaction degree of the medium was observed on the 5th, 10d, 20d and 30d respectively. Before observation, the test tube needs to be cooled for 20-30 minutes or rinsed with tap water for 30 minutes to observe the liquefaction degree of the medium.

⑤ Cellulose decomposition:

Submerge one end of the filter paper strip in the liquid medium, after sterilization, inoculate the strain to be tested on the filter paper above the liquid level, and observe whether the filter paper strip is decomposed one month later.

⑥ Nitrate reduction:

The strains to be tested were inoculated in nitrate-reducing medium, and cultured at 28°C for 7 and 14 days, and the culture medium without bacteria was used as a control. Add a little culture solution that has been cultivated for 7 days and 14 days respectively in the test tube, add a drop of solution A and solution B, and add dropwise the same for the control. When the solution turns pink, rose red, orange or brown, etc., it is positive for nitrate reduction; if no red appears, add 1 or 2 drops of diphenylamine reagent, if it turns blue, the reduction is negative; if not Blue is still treated as positive.

(2) Single carbon source utilization experiment

In the identification of actinomycetes, one of the important inspection indicators is the utilization of carbon sources. Different actinomycetes have different carbon source utilization capabilities such as sugars, alcohols, organic acids, and fatty acids. Carbon sources used in the experiment: D-fructose, xylose, rhamnose, arabinose, raffinose, melezitose, anhydrous lactose, D-galactose, α-lactose, D-trehalose, D-mannose Sugar, D-ribose, inositol, sorbitol, mannitol, salicin, and soluble starch were added to Pugo's basal medium at a carbon source concentration of 1%. And inoculate the strain to be tested, and culture at a constant temperature of 28°C for 7-14 days. The strain inoculated with the basal medium without adding any carbon source is used as a blank control, and the growth of the strain is observed. If it can grow, it indicates that the strain can utilize this carbon source; if it cannot grow, it indicates that the strain cannot utilize this carbon source.

(3) Single nitrogen source utilization experiment

Nitrogen sources used in the test: histidine, methionine, serine, oxalic acid, glycine, hydroxyproline, phenylalanine, glutamic acid, cysteine, arginine, valine, four Ammonium molybdate hydrate, ammonium acetate, ammonium nitrate, and ammonium sulfate are added to the basal medium at a concentration of 0.5%. Inoculate the strains, culture at a constant temperature of 28°C for 7-14 days, use the strains inoculated with the basal medium without any nitrogen source as the blank control, and observe the growth of the strains. If it can grow, it means that the strain can use the nitrogen source; if it cannot grow, it means that the strain cannot use the nitrogen source.

(4) Determination of some other physiological and biochemical indicators

①Temperature tolerance test:

After inoculating the strain to be tested in the same culture medium, culture them at 20°C, 24°C, 28°C, 32°C, and 36°C for 7-14 days under the condition that other culture conditions are the same, observe and record the growth of the colony conditions to determine the optimum temperature for strain growth.

②pH tolerance test:

The strains to be tested were inoculated in the liquid medium with pH values of 4, 5, 6, 7, 8, 9, and 10, to ensure that other culture conditions were consistent, cultured at 28°C, and observed every other week. Observed until four weeks. Observe and record the growth of the strain each time to determine the optimum pH for the growth of the strain.

③Salt tolerance test:

The strains to be tested were respectively inoculated on different concentrations of NaCl (1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%) medium, and other nutrient components of the medium were all the same. Culture at 28°C, 7 days as an observation period, observe for 4 weeks, and record whether it can grow, so as to determine the upper and lower limit concentrations of NaCl that the strain can tolerate.

④ Hydrogen sulfide production experiment:

Inoculate the strain to be tested on a hydrogen sulfide medium containing a certain proportion of ammonium ions, and culture it at 28°C for 2 weeks. If the medium is black, hydrogen sulfide is produced; if there is no black, there is no hydrogen sulfide production. 2.1.7 Molecular biological identification of antagonistic strains

(1) Extraction of Actinomycetes Genomic DNA

Total DNA was extracted using Bioteke's Bacterial Genomic DNA Rapid Extraction Kit (DP1301, Beijing Bioteke Biotechnology Co., Ltd., China).

(2) Sequencing and analysis of 16S rDNA

①PCR amplification of 16S rDNA:

Genomic DNA of actinomycetes was used as a template, and the general primers 27F and 1492R were used for PCR amplification. The primer sequences are as follows: upstream primer 27F (5'AGAG TTTG ATCC TGGC TCAG 3'), downstream primer 1492R (5'TACG GCTACCTTGTTACGAC TT 3'). The specific reaction system is shown in Table 7, and the reaction procedures (Zhou Junping, 2014; Hao Feifei, 2015; Na Yua, 2014) are shown in Table 8.

Table 7 PCR reaction system of 16S rDNA gene

Table 8 PCR amplification reaction conditions of 16S rDNA gene

② Electrophoresis detection of PCR products:

After the PCR reaction, take 5 μL of the PCR amplification product and perform electrophoresis detection on the PCR product of the strain on a 1% agarose gel, and determine whether the connection is successful according to the length of the target fragment.

③Sequencing and building a phylogenetic tree:

The PCR product of the strain was sent to Huada Gene Company for sequence determination. The measured gene sequences were compared using BLAST software, and compared with the known 16S rDNA in GenBank and EzBioCloud databases. The sequences with high homology were found for multiple matching alignment analysis, and the Neighbor-Joining method was used for clustering analysis and phylogenetic tree construction using MEGA5.1 software (Na Yua, 2014; Jianghua, 2015).

2.2 Evaluation of antibacterial activity of antagonistic strains

2.2.1 Determination of plate confrontation broad-spectrum antibacterial activity

The plate confrontation method was used to measure the broad-spectrum of the strains: use a 5mm hole punch to take the purified cakes of two plant pathogenic bacteria, inoculate them in the center of the PDA plate, and place them in four places 2.5cm away from the pathogenic bacteria cakes. A small amount of bacteria to be tested was inoculated on the spot, and the petri dish only inoculated with pathogenic bacteria was used as the blank control group, and each treatment was repeated 3 times. After culturing in the incubator for 4-7 days, the colony growth diameter of the tested pathogenic bacteria was measured by the cross method, and the antibacterial rate was calculated according to the following formula (Xie Ying, 2011; Xia Longsun, 2013):

Colony diameter (mm) = average value of measured colony diameter - 5.0

2.2.2 Determination of antibacterial activity of secondary metabolites against pathogenic bacteria

The strains were cultured in SLM liquid medium for 7 days, added absolute ethanol and shaken for 10 days, vacuum-filtered with 5 layers of filter paper, and the secondary metabolites of the strains were collected as samples. Using the antibacterial paper method (Xu Shuyun et al., 2003; Wellman-Labadie et al., 2010), take the sterilized culture medium and pour it on a plate. After the plate is solidified, use a puncher to make a filter paper sheet with a diameter of 9 mm. The filter paper was sterilized by dry heat for 2 hours, soaked in the sample for 30 minutes in an aseptic operating table, took out and evaporated the solvent, and pasted the filter paper flat on the above-mentioned drug-containing plate, and at the same time inoculated the indicator bacteria in the center of the culture medium. Finally, all culture dishes were placed in a constant temperature incubator at 28°C for cultivation, observed and recorded and photographed every day, and three parallels were made for each group, and the average diameter of the inhibition zone was calculated.

2.3 Preparation of strain fermentation broth

Fermentation broth preparation: measured by Haikou Academy of Agricultural Sciences, the nitrogen content of soybean meal is 7.0%, the C/N is 6.76, and the carbon content of molasses is 20.27%. In this experiment, based on the carbon-to-nitrogen ratio of soybean meal and the carbon content of molasses, the carbon-to-nitrogen ratio of the substrate was adjusted to 25:1, and the total amount of molasses and soybean meal was 250g, that is, 34.25g of soybean meal, 215.75g of molasses, and 1000mL of sterile water to prepare molasses And cake fertilizer mixture, put into a 5L Erlenmeyer flask for testing.

Bacterial flora construction: Configure YE liquid medium, pack in 250mL Erlenmeyer flasks, sterilize at 121°C for 20 minutes, add fresh strains to be tested after cooling, shake at 180rpm/min at 28-30°C After 3 days of bed culture, 5% of the inoculum was inserted into the newly prepared fermentation nutrient solution, and the fermentation was carried out for 9 days at 28-30° C. and 180 rpm/min on a shaking table for testing.

2.4 Pot experiment of antagonistic actinomycetes on banana fusarium wilt

The pot experiment was carried out in the greenhouse of the Institute of Tropical Biotechnology of the Chinese Academy of Tropical Agricultural Sciences from May to July 2017. The conditions of the greenhouse were controlled as follows: temperature around 28°C, humidity 70%, and natural light. There were 4 treatments in the experiment, CK1: no pathogenic bacteria, and sterile water; CK2: pathogenic bacteria, and sterile water; CK3: pathogenic bacteria, and dikexone; A: pathogenic bacteria, and no bacteria fermented liquid; AB : Inoculate pathogenic bacteria and apply strain XJC-HDM11 fermentation broth.

Inoculation by soaking fungus in root wounds. Select banana seedlings with the same growth and 5-6 leaves, cut off the second main root, soak in the pathogen spore suspension with a concentration of 10 ⁵ /mL for 30 minutes, and then transplant them into plastic pots with a soil content of 700g 20 mL of pathogenic bacteria suspension was poured in the rhizosphere soil of banana seedlings, and each treatment was repeated 3 times, with 20 banana seedlings in each repetition.

Disease index statistics. The graded counting method was used to observe the incidence after inoculation of pathogenic bacteria, and the disease index was counted starting from the first banana seedling infected. After all the banana seedlings were infected, 100-fold diluted fermentation broth was applied according to different treatments, 200 mL per plant, and the same amount of water was applied to CK; the fermentation broth and water were repeated every 15 days, for a total of 3 times.

During the test period, other management measures were the same for each treatment. On the 60th day after banana transplantation, the number of yellowed leaves and normal leaves per banana plant in each treatment was recorded, and the disease index and control effect were calculated. Banana Fusarium wilt grading standard: Grade 0, healthy plants; Grade 1, 25% yellowed diseased leaves; Grade 3, 25%-50% yellowed diseased leaves; Grade 5, 50%-90% yellowed diseased leaves Diseased leaves; grade 7, all leaves turn yellow and the plant dies. Calculation of banana disease index and disease prevention effect:

2.5 Investigation and determination of physiological indicators of banana plants

After the banana seedlings were transplanted on 0d, 15d, 30d, 45d, and 60d, the chlorophyll content of the second fully expanded leaf at the top of each treatment was measured by ethanol extraction method; on the 60th day, the leaf area, root length and Diameter, plant height, pseudostem girth, aboveground biomass, root biomass.

3 Results and Analysis

3.1 Screening and morphological characteristics of actinomycetes

The strains isolated and purified by plate coating were deduplicated according to their colony shape and color on the purified medium, and after the initial screening by the plate confrontation culture method and the re-screening by the Oxford cup method, the strain with the largest inhibition zone was obtained. 1 strain of actinomycetes; numbered XJC-HDM11.

The culture characteristic of table 9 bacterial strain XJC-HDM11 on 6 kinds of media

3.2 Physiological and biochemical characteristics of the strain

Table 10 Some physiological and biochemical characteristics of strain XJC-HDM11

+: the result is positive; -: the result is negative.

3.3 Phylogenetic characteristics of strains

The total DNA of the strain XJC-HDM11 was extracted, the 16S rDNA sequence of about 1.5kb was obtained by PCR amplification, and its sequence was obtained by sequencing. The sequence information was submitted to EzTaxon for gene sequence similarity search, and a total of 9 strains were homologous to the strain XJC-HDM11 The sequence information of the highest and named model bacteria was analyzed for phylogenetic analysis, and a phylogenetic tree was constructed (Figure 2). It can be seen from the figure that XJC-HDM11 and Streptomyces are clustered together, and the homology of 16S rDNA sequence is in the range of 98.95% to 99.56%. Among them, the homology with Streptomyces yanii is the highest at 99.42%.

Combined with the results of morphological characteristics, physiological and biochemical characteristics and 16s DNA molecular sequence analysis, the strain XJC-HDM11 was preliminarily identified as Streptomyces yanii.

3.4 Evaluation of antibacterial activity of strain XJC-HDM11

3.4.1 Antagonistic effect of plate confrontation on pathogenic bacteria

The inhibitory effect of table 11 bacterial strain XJC-HDM11 to pathogenic fungus

The data in the table are mean ± standard deviation. Different small letters in the same column indicate significant difference at P<0.05 level by Duncan's new multiple range test.

3.4.2 Antibacterial activity of the crude extract of strain XJC-HDM11 on the growth of pathogenic mycelium

The inhibitory effect of the crude extract of table 12 bacterial strain XJC-HDM11 to pathogenic fungi

3.4.3 Control effect of fermentation broth of strain XJC-HDM11 on banana Fusarium wilt

In the pot experiment, the incidence of banana plants in each treatment is shown in Table 12. The results showed that there was no significant difference in the disease index and control effect of different treatments of banana seedling Fusarium wilt. No pathogenic bacteria were picked up, and the banana seedlings with sterile water (CK1) had no symptoms and grew well. Inoculated with pathogenic bacteria, the disease index of the application of clear water (CK2) and the application of fermented liquid (A) without adding bacteria were all high, 84.38 and 70.45 respectively; while the disease index of inoculated antagonistic bacteria XJC-HDM11 fermented liquid (AB) was 10.23, The prevention and control effect is as high as 83.12%.

Table 13 Pot control effect of antagonistic actinomycetes XJC-HDM11 on banana Fusarium wilt

The data in the table are mean ± standard deviation. Different small letters after the data in the same column indicate significant difference at P<0.05 level by Duncan's new multiple range test.

3.4.4 Effect of the fermentation broth of strain XJC-HDM11 on the chlorophyll content of banana leaves

As shown in Figure 3, for the five treatments, the chlorophyll content of bananas increased with the increase of transplanting time, the chlorophyll content of CK1 continued to increase, the chlorophyll content of CK2 showed a decreasing trend, and the CK3, A, and AB treatments all decreased first and then increased, while The lowest critical point of AB treatment was higher than that of CK3 and A treatment, and the upward trend was also significantly higher than that of the other three treatments. Because the chlorophyll content was negatively correlated with the incidence rate, the control effect of CK1 treatment was the worst. All 20 banana seedlings were infected, and the disease index continued to increase with time, and the chlorophyll content of the treatment also decreased; CK1 treatment was not inoculated with pathogenic bacteria. After transplanting, the banana seedlings grew healthy, but the chlorophyll content only showed a slow growth trend, the highest was 0.99mg/g; CK3 and A treatment, although the chlorophyll showed a growth trend in the later period, due to the disease, the growth point was always lower than that of the untreated banana seedlings. The diseased blank control CK1 was 0.81mg/g and 0.68mg/g respectively; on the contrary, due to the presence of antibiotics, the disease index of banana seedlings decreased in the later stage of AB treatment, and the disease prevention effect was remarkable. The chlorophyll showed a linear and rapid growth trend, reaching 1.28 at 60 days mg/g, significantly higher than other treatments. This indicated that the fermentation culture of antagonistic bacteria XJC-HDM11 was beneficial to increase the chlorophyll content of banana.

2.4.5 Effect of the fermentation broth of strain XJC-HDM11 on the growth of banana plants

As can be seen from Table 14, the banana seedlings treated with Dikesong (CK3) had leaf area (1155.42cm ² ), root length (1036.92cm), root diameter (0.78mm), plant height (46.22cm), stem diameter ( 4.97cm) was significantly higher than that of the other three treatments. The physiological indexes of A treatment were only higher than those of CK2, which were completely infected, and were significantly lower than those of AB, CK3 and CK1. Significantly smaller, the plant is short and the leaves are yellowish and narrow. However, the leaf area (1398.13cm ² ), root length (1354.87cm), root diameter (1.11mm), plant height (53.76cm) and stem diameter (6.19cm) of banana seedlings treated with the antagonistic strain XJC-HDM11 fermentation broth were significantly Higher than other treatments, it shows that the antagonistic bacteria XJC-HDM11 fermentation liquid not only has disease-resistant activity, but also has a growth-promoting effect on banana seedlings, which can promote plant leaf area, enhance photosynthesis, increase root growth, and promote transpiration, thereby promoting banana plant growth. grow, increase yield.

Table 14 Antagonistic actinomycetes XJC-HDM11 on the growth of banana plants

The data in the table are mean ± standard deviation. Different small letters after the data in the same column indicate significant difference at P<0.05 level by Duncan's new multiple range test.

2.4.6 Effect of the fermentation broth of strain XJC-HDM11 on the biomass of banana plants

It can be seen from Figure 4 and Figure 5 that the fresh weight and dry weight of the aerial parts of banana seedlings in the five treatments are AB>CK1>CK3>A>CK2, and the AB treatment is significantly higher than the other four treatments, the fresh weight of the aerial part It is 48.20g, the dry weight is 4.34g, and the water content is 90.99%. As for the fresh weight of the underground part, there was no significant difference between AB treatment and CK1, but it was significantly higher than the other three treatments, while CK3 was significantly higher than CK1 and CK2, indicating that CK3 treatment had a certain control effect, but not as good as AB treatment; Dry weight, AB treatment was significantly higher than other treatments, CK1 treatment and CK3 treatment had no significant difference, but was significantly higher than CK2 and A treatment. After the treatment of AB fermentation liquid, the dry matter accumulation also increased significantly, which was higher than that of normal growing banana seedlings, which indicated that the XJC-HDM11 fermentation liquid had a significant effect on promoting the growth and biomass accumulation of bananas.

The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention shall fall within the scope of the present invention.

Claims (9)

1. a kind of actinomyces, it is characterised in that it is Yan Shi streptomycete XJC-HDM11, and deposit number is CCTCC NO：M 2017493。

2. the zymotic fluid of the actinomyces described in claim 1 or the ethanol extract of the filtered fluid of zymotic fluid or zymotic fluid.

3. the filtering of the zymotic fluid or zymotic fluid of actinomyces as claimed in claim 1 or actinomyces as claimed in claim 2 Application of the ethanol extract of liquid or zymotic fluid in antagonism banana blight bacteria.

4. application as claimed in claim 3, it is characterised in that the actinomyces be applied to antagonism banana blight bacteria 1, And/or banana blight bacteria 4.

5. the filtering of the zymotic fluid or zymotic fluid of actinomyces as claimed in claim 1 or actinomyces as claimed in claim 2 Application of the ethanol extract of liquid or zymotic fluid in banana blight is prevented.

6. the filtering of the zymotic fluid or zymotic fluid of actinomyces as claimed in claim 1 or actinomyces as claimed in claim 2 Application of the ethanol extract of liquid or zymotic fluid in the chlorophyll content for improving banana.

7. the filtering of the zymotic fluid or zymotic fluid of actinomyces as claimed in claim 1 or actinomyces as claimed in claim 2 Application of the ethanol extract of liquid or zymotic fluid in banana plant growth is promoted.

8. the filtering of the zymotic fluid or zymotic fluid of actinomyces as claimed in claim 1 or actinomyces as claimed in claim 2 Application of the ethanol extract of liquid or zymotic fluid in banana biomass accumulation is promoted.

9. a kind of microbial inoculum, it is characterised in that contain the actinomyces described in claim 1.