阅读说明：本技术 一种yhg-1链霉菌在抑制植物病原真菌及促香蕉生长的应用 (Application of Streptomyces YHG-1 in inhibiting plant pathogenic fungi and promoting banana growth ) 是由 井涛 臧小平 周登博 云天艳 王尉 谢江辉 陈宇丰 于 2021-08-30 设计创作，主要内容包括：本发明提供一种YHG-1链霉菌在抑制植物病原真菌及促香蕉生长的应用,YHG-1链霉菌对植物病原菌具有明显的拮抗作用,广谱性好,抑菌率达69％,并且YHG-1链霉菌发酵液既具有明显的抗病活性,对香蕉枯萎病菌的防治效果高达88.95％,又可香蕉苗具有明显的促生作用,以香蕉苗根系的生长最为显著,具有很好的开发应用前景。(The invention provides an application of Streptomyces YHG-1 in inhibiting plant pathogenic fungi and promoting banana growth, the Streptomyces YHG-1 has obvious antagonistic action on plant pathogenic fungi, the broad spectrum is good, the bacteriostasis rate reaches 69%, and the fermentation broth of the Streptomyces YHG-1 has obvious disease resistance activity, the prevention and treatment effect on banana wilt bacteria reaches 88.95%, and the fermentation broth of the Streptomyces YHG-1 has obvious growth promoting effect on banana seedlings, so that the growth of the banana seedling root system is most obvious, and the development and application prospect is good.)

1. An application of Streptomyces YHG-1 in inhibiting plant pathogenic fungi is disclosed.

2. The use of streptomyces YHG-1 as claimed in claim 1 for the inhibition of phytopathogenic fungi, characterized in that: the preservation number of the Streptomyces YHG-1 is CCTCC NO: m2021007.

3. The use of streptomyces YHG-1 as claimed in claim 1 for the inhibition of phytopathogenic fungi, characterized in that: the Streptomyces YHG-1 is applied to inhibition of litchi colletotrichum, mango colletotrichum, banana colletotrichum, pepper colletotrichum, cabbage colletotrichum, strawberry colletotrichum, botrytis cinerea, banana vascular wilt bacterium No. 4 microspecies, banana vascular wilt bacterium No. 1 microspecies, banana tree canker bacteria, banana large gray spot bacteria and banana long spot bacteria.

4. The use of streptomyces YHG-1 as claimed in claim 1 for the inhibition of phytopathogenic fungi, characterized in that: the YHG-1 streptomycete is applied to preparation of YHG-1 streptomycete fermentation liquor, and the YHG-1 streptomycete fermentation liquor has an antagonistic effect on plant pathogenic fungi.

5. The use of Streptomyces YHG-1 as claimed in claim 4 for the inhibition of phytopathogenic fungi, wherein: the YHG-1 streptomyces fermentation liquid has an antagonistic effect on banana fusarium wilt.

6. Application of Streptomyces YHG-1 in promoting banana growth.

7. The use of Streptomyces YHG-1 as claimed in claim 6 for promoting banana growth, wherein: the YHG-1 streptomycete is applied to preparation of the YHG-1 streptomycete fermentation liquor, and the YHG-1 streptomycete fermentation liquor has a promoting effect on banana growth.

8. The use of Streptomyces YHG-1 as claimed in claim 6 for promoting banana growth, wherein: the YHG-1 streptomyces fermentation liquid is applied to promoting the growth of banana seedling root systems.

9. The use according to claim 4 or claim 7, wherein: the preparation method of the YHG-1 streptomycete fermentation liquid comprises the following steps:

(1) preparing a fermentation nutrient solution: taking bean pulp with the nitrogen content of 7.0 percent and molasses with the carbon content of 20.27 percent, and adding water to prepare fermentation nutrient solution with the carbon-nitrogen ratio of 25: 1;

(2) preparing YE liquid culture medium: adding 4g/L of ddH2O, mixing with 4g/L of glucose, 4g/L of yeast powder and 10g/L of maltol, adjusting pH to 7.2-7.4 to obtain YE liquid culture medium, and sterilizing;

(3) preparing a YHG-1 streptomyces seed liquid: inoculating YHG-1 streptomycete strains into YE liquid culture medium, and performing shaking table culture at 160-200 rpm/min for 2-4 days at 28-30 ℃ to obtain YHG-1 streptomycete seed liquid;

(4) inoculating the YHG-1 streptomycete seed liquid into a fermentation nutrient solution according to the inoculation amount of 5 wt%, and culturing for 8-10 days under the conditions of a shaker at 28-30 ℃ and 160-200 rpm/min to obtain the YHG-1 streptomycete fermentation liquid.

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to application of Streptomyces YHG-1 in inhibiting plant pathogenic fungi and promoting banana growth.

Background

The banana is a perennial herb of Musa of Musaceae, and is one of the main fruits of tropical and subtropical zone. China is a large banana producing country and a large banana consuming country, the banana planting area reaches 40 million hectares, and the consumption is the first in the global ranking. The banana cultivation area is wide, so that outbreaks of various plant diseases and insect pests are caused, the plant diseases mainly come from three major types, namely bacteria, fungi and viruses, and the plant diseases caused by the fungi account for about 70-80% of the plant diseases, such as banana vascular wilt, banana anthracnose, banana gray leaf spot and the like, wherein the banana vascular wilt is the most serious, and once the banana vascular wilt explodes in a large scale, the plant diseases bring destructive attack to local banana cultivation. With the environmental protection, green food and agricultural sustainable development, the research and development of antimicrobial agents of microbial origin become one of the main research hotspots.

With the intensive research on streptomyces, through continuous optimization and improvement on a separation and culture method, more and more streptomyces are separated, the streptomyces are considered as main sources for generating various antibiotics, and are gradually applied to the fields of agriculture, medicine and the like due to genetic diversity, but the defects of weak antagonistic capability of strains on pathogenic bacteria, low broad spectrum and the like exist, so that the streptomyces capable of inhibiting plant pathogenic fungi diseases in a broad spectrum are found, the streptomyces can be effectively applied to cultivation and planting of crops such as bananas, and the streptomyces has important significance on the wide application of microbial antibacterial agents in the aspects of plant growth and disease control.

Disclosure of Invention

Therefore, the invention provides an application of Streptomyces YHG-1 in inhibiting plant pathogenic fungi and promoting banana growth.

The technical scheme of the invention is realized as follows:

an application of Streptomyces YHG-1 in inhibiting plant pathogenic fungi is disclosed.

The preservation number of the Streptomyces YHG-1 sp.Nov.YHG-1 in China center for type culture Collection is CCTCC NO: m2021007; the preservation time is 2021 year, 1 month and 19 days. The YHG-1 streptomyces species has a growth pH range of 5-8, a growth temperature of 25-30 ℃, can not grow on a culture medium with a NaCl content of more than 7 percent, and has broad-spectrum bacteriostatic activity.

Further explaining, the Streptomyces YHG-1 can be applied to the inhibition of litchi colletotrichum, mango colletotrichum, banana colletotrichum, pepper colletotrichum, cabbage colletotrichum, strawberry colletotrichum, botrytis cinerea, banana vascular wilt bacteria No. 4 microspecies, banana vascular wilt bacteria No. 1 microspecies, banana tree canker bacteria, banana gray spot bacteria and banana physalospora piricola.

Further illustrates the application of the Streptomyces YHG-1 in the preparation of the Streptomyces YHG-1 fermentation broth, wherein the Streptomyces YHG-1 fermentation broth has an antagonistic effect on plant pathogenic fungi.

Further illustrates that the YHG-1 streptomyces fermentation broth has antagonistic action on banana fusarium wilt.

Application of Streptomyces YHG-1 in promoting banana growth.

Further illustrates that the YHG-1 streptomyces is applied to preparation of the YHG-1 streptomyces fermentation liquor, and the YHG-1 streptomyces fermentation liquor has a promoting effect on banana growth.

Further illustrates the application of the YHG-1 streptomyces fermentation broth in promoting the growth of banana seedling root systems.

The preparation method of the YHG-1 streptomycete fermentation liquid comprises the following steps:

(1) preparing a fermentation nutrient solution: taking bean pulp with the nitrogen content of 7.0 percent and molasses with the carbon content of 20.27 percent, and adding water to prepare fermentation nutrient solution with the carbon-nitrogen ratio of 25: 1;

(2) preparing YE liquid culture medium: adding 4g/L of ddH2O, mixing with 4g/L of glucose, 4g/L of yeast powder and 10g/L of maltol, adjusting pH to 7.2-7.4 to obtain YE liquid culture medium, and sterilizing;

(3) preparing a YHG-1 streptomyces seed liquid: inoculating YHG-1 streptomycete strains into YE liquid culture medium, and performing shaking table culture at 160-200 rpm/min for 2-4 days at 28-30 ℃ to obtain YHG-1 streptomycete seed liquid;

(4) inoculating the YHG-1 streptomycete seed liquid into a fermentation nutrient solution according to the inoculation amount of 5 wt%, and culturing for 8-10 days under the conditions of a shaker at 28-30 ℃ and 160-200 rpm/min to obtain the YHG-1 streptomycete fermentation liquid.

Compared with the prior art, the invention has the beneficial effects that: the Streptomyces YHG-1 provided by the invention can be applied to plant pathogenic bacteria for inhibiting litchi colletotrichum, mango colletotrichum, banana colletotrichum, pepper colletotrichum, cabbage colletotrichum, strawberry colletotrichum, botrytis cinerea, banana vascular wilt 4 subspecies, banana vascular wilt 1 subspecies, banana tree canker, banana large gray spot and banana physalospora piricola, has good broad spectrum property, and has a maximum inhibition rate of 69%.

Drawings

FIG. 1 is a graph showing the control effect of different treatments on banana vascular wilt in accordance with the present invention;

FIG. 2 is a line graph showing the effect of different treatments on chlorophyll content of banana leaves according to examples of the present invention;

FIG. 3 is a bar graph of the impact of different treatments on fresh weight of aerial and underground portions of bananas in accordance with an embodiment of the invention;

FIG. 4 is a bar graph of the effect of different treatments on the dry weight of the aerial and underground portions of bananas in accordance with an embodiment of the invention;

Detailed Description

In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.

The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.

The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.

Example 1-YHG-1 measurement of bacteriostatic Activity of Streptomyces strains

The invention adopts a flat plate confronting method to perform broad-spectrum determination on the YHG-1 streptomyces strain: a5 mm puncher is used for taking a purified bacterial cake of 12 plant pathogens, the bacterial cake is inoculated to the center of a PDA (personal digital assistant) plate, a small amount of YHG-1 streptomycete is inoculated to four points 2.5cm away from the pathogen bacterial cake, a culture dish only inoculated with the pathogens is used as a blank control group, and each treatment is repeated for 3 times.

After culturing for 4-7 days in an incubator, measuring the colony growth diameter of pathogenic bacteria to be tested by adopting a cross method, counting the bacteriostasis rate (Xiexing, 2011; Xia Longsun, 2013) according to the following formula,

colony diameter (mm) — average value of colony diameters measured-5.0

The bacteriostatic effect of the Streptomyces YHG-1 strain on plant pathogenic fungi is shown in the following table 1:

example 2-preparation of a Streptomyces YHG-1 fermentation broth comprising the following steps:

(1) preparing a fermentation nutrient solution: taking bean pulp with the nitrogen content of 7.0 percent and molasses with the carbon content of 20.27 percent measured by an analysis test center of the Chinese tropical agricultural academy of sciences, adding water to prepare a fermentation nutrient solution with the carbon-nitrogen ratio of 25:1, wherein the total amount of the molasses and the bean pulp is 250g, namely 34.25g of the bean pulp, 215.75g of the molasses and 1000mL of sterile water for later use;

(2) preparing YE liquid culture medium: adding 4g/L of ddH2O, mixing with glucose, 4g/L of yeast powder and 10g/L of maltol, adjusting pH to 7.2-7.4 to obtain YE liquid culture medium, and sterilizing at 121 deg.C for 20 min;

(3) preparing a YHG-1 streptomyces seed liquid: inoculating YHG-1 streptomycete strains into YE liquid culture medium, and carrying out shake cultivation at the temperature of 28-30 ℃ and the rpm of 180/min for 3 days to obtain YHG-1 streptomycete seed liquid;

(4) inoculating the YHG-1 streptomycete seed liquid into a fermentation nutrient solution according to the inoculation amount of 5 wt%, and culturing for 9 days at the temperature of 28-30 ℃ under the condition of a shaking table at 180rpm/min to obtain the YHG-1 streptomycete fermentation liquid.

Example 3 prevention and control experiment of Streptomyces YHG-1 fermentation broth for Banana wilt disease

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 with 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, and applying fenaminosulf;

a: inoculating pathogenic bacteria, and applying fermented nutrient solution without adding bacteria;

AB: inoculating the pathogenic bacteria, and applying fermentation liquor containing the strain YHG-1.

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:

disease index [ ∑ (number of disease strains at each stage × number of disease stages) ]/total number of investigated strains × number of highest disease stages × 100

Disease prevention effect is (contrast disease index-treatment disease index)/contrast disease index x 100%

(1) According to the disease index, the severity of banana wilt can be directly reflected, the disease index and the prevention and treatment effect of the banana under the potting condition are shown in the table 2, and the banana is healthily grown and has no disease when being treated by CK1 (without pathogenic bacteria); the rest treatments connected with the pathogenic bacteria have different degrees of disease symptoms, the disease index difference between different treatments is obvious, the disease index is shown as CK2 more than A more than CK3 more than AB, the CK2 treatment causes all diseases, the disease index is up to 82.97, the A treatment is less than 71.09, which is obviously higher than that of the treatment with the fenaminosulf agent; the banana treated by AB (strain YHG-1 fermentation liquor) has the lightest morbidity, the disease index is only 9.17, the control effect reaches 88.95 percent, the banana vascular wilt control effect is obvious (figure 1), the control effect of the banana vascular wilt without adding the strain YHG-1 is very little and is far lower than that of a common medicament, and after adding the inoculation strain YHG-1, the control effect is obvious, the influence of pathogenic bacteria can be reduced to the minimum, and the banana growth can be promoted.

TABLE 2 potted plant control effect of antagonistic actinomycetes YHG-1 on banana fusarium oxysporum

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.

Example 4 Effect of Streptomyces YHG-1 fermentation broth on chlorophyll content of Banana leaves

The chlorophyll content of banana leaves is inversely related to the incidence rate of the blight, the more serious the blight occurs, the more yellow the banana leaves are, and the lower the chlorophyll content is. As can be seen from FIG. 2, the chlorophyll content of the banana leaves treated by CK1 (non-pathogenic bacteria) is gradually increased with the increase of the transplanting time, and reaches a maximum value of 0.98mg/g FW in about 40 days; treating 20 bananas with CK2 (only inoculated with pathogenic bacteria) to prevent diseases, wherein the control effect is the worst, the chlorophyll content of the banana leaves is continuously reduced, and the initial content is about 0.72mg/g FW and is reduced to 0.2mg/g FW; the chlorophyll content of the other 3 treated banana leaves showed a tendency of decreasing and then increasing, except that the chlorophyll content of CK3 (fenaminosulf) treatment and A (without the fermentation nutrient solution of the strain YHG-1) treatment were significantly decreased within 20 days, the chlorophyll content increased to 0.88 and 0.75mg/g FW at 40 days, which was always lower than that of CK1, and the chlorophyll content of the A treatment was nearly recovered to the initial level; and the chlorophyll content of the banana treated by AB (containing the strain YHG-1 fermentation liquor) is only slightly reduced in about 10 days, and then the chlorophyll content is in a trend of rapidly increasing, and at 40 days, the chlorophyll content reaches the highest 1.18mg/g FW which is obviously higher than that of other treatments, and the increase rate is also higher than that of CK1 treatment, so that the application of the strain YHG-1 fermentation liquor enables the treated banana to completely get rid of the influence of blight, and the improvement of the chlorophyll content of the banana is more favorably improved.

Example 5 Effect of Streptomyces YHG-1 fermentation on Banana plant growth

The overground part and root system of the bananas treated by each treatment were different (Table 3). The integral growth of the overground part and the underground part of the banana is expressed by AB & gt CK1 & gt CK3 & gt A & gt CK2 treatment. The leaf area (369 cm) of each physiological index of bananas treated only by pathogenic bacteria (CK2)²) The plant height (23.85cm), stem thickness (3.84cm), root length (444cm) and root diameter (0.43mm) are all obviously lower than those of other treatments, the banana leaves are all yellow and narrow, and the plants are thin and weak; the banana is thicker and stronger in normal growth aroma (CK1), the root system is more developed, the stem thickness (4.87cm) and the root diameter (0.7mm) are obviously lower than those of the dikesong (CK3) and higher than those of the fermented liquid (A) without the bacterial strain YHG-1; after the strain YHG-1 fermentation broth (AB) is applied, the banana grows best and the area of the banana leaf is (1408 cm)²) Stem thickness (6.21cm), root length (1520cm) and root diameter (1.23mm) were all significantly higher than other treatments, 18.12% higher than normal growing (CK1) bananas respectively27.52%, 40.22% and 75.71%.

Therefore, the inoculated antibacterial YHG-1 fermentation liquor has good disease resistance activity on banana wilt and also has obvious growth promotion effect on banana seedlings, particularly on the growth of the root systems of the banana seedlings, the effect is more obvious, the length of the root systems is increased by more than 40%, and the diameter of the root systems is increased by more than 70%. The root system is crucial to the growth and development of plants, the antibacterial YHG-1 fermentation liquor is inoculated, the harm of fusarium oxysporum is reduced, meanwhile, the growth of the root system is remarkably promoted, the stress resistance of the banana is further improved, and a good physiological basis is laid for the healthy growth, high quality and high yield of the banana.

TABLE 3 growth Effect of antagonistic Actinomycetes YHG-1 on Banana plants

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.

Example 6 Effect of Strain YHG-1 fermentation broth on Banana plant Biomass

As can be seen from FIGS. 3 and 4, the fresh weight and the dry weight of the aerial parts of the banana seedlings are AB > CK1 > CK3 > A > CK2, and the difference between different treatments is obvious; the fresh weight and dry weight of each treated underground part are basically consistent with the expression of the overground part, and slightly different are that the fresh weight and dry weight of the CK3 and A treated underground parts have no significant difference.

Therefore, the biomass of the overground part of the banana is increased to be highest by 48.82 g/strain when the AB (strain YHG-1 fermentation liquor) is used for treating the overground part of the banana, and is increased by 27.60% compared with CK1 (banana under normal conditions); under the condition of pathogenic bacteria stress, the biomass of banana seedlings is respectively increased by 73.34 percent and 37.75 percent by treating CK3 (fenaminosulf) and A (without adding a strain YHG-1 fermentation nutrient solution) compared with CK2 (only inoculating pathogenic bacteria), and the biomass of the banana seedlings is obviously lower than that of CK1 by treating the CK3 (without adding the strain YHG-1 fermentation nutrient solution), so that the growth condition of bananas can be obviously improved by treating CK3 and A under the condition of pathogenic bacteria stress, but normal growth of bananas cannot be completely recovered, and the growth promoting effect of the treatment A is far obviously lower than that of medicament treatment. Therefore, the influence of pathogenic bacteria on bananas cannot be completely counteracted no matter the banana is treated by the medicament or not added with the strain YHG-1 fermentation nutrient solution, and the addition of the strain YHG-1 fermentation broth AB can not only completely eliminate the adverse effect of the pathogenic bacteria on bananas, but is even obviously superior to normal growth of bananas, and has the most obvious growth promoting effect on bananas.

Example 7 Effect of different Streptomyces YHG-1 fermentation broths on Banana plant growth

The preparation method of the fermentation broth of the YHG-1 Streptomyces is as in example 2, wherein the carbon-nitrogen ratio in the fermentation nutrient solution is adjusted to 10:1, the fermentation broth is used for inoculating the seed solution of the YHG-1 Streptomyces with the inoculation amount of 5 wt%, and the seed solution is cultured for 9 days at 28-30 ℃ under the condition of a shaking table at 180rpm/min to obtain the fermentation broth of the YHG-1 Streptomyces. The experimental results of the overground and root system growth of bananas show that certain difference exists in the influence on the growth of bananas, wherein the growth difference on the root length and the root diameter of banana seedlings is most obvious, and the root length and the root diameter ratio AB of bananas are respectively 6.5% and 10.2% lower, which shows that the YHG-1 streptomycete fermentation liquor prepared by nutrient solutions with different carbon-nitrogen ratios has obvious difference on the growth action of the root systems of bananas.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.