阅读说明：本技术 淀粉酶产色链霉菌代谢产物在防治浙麦冬黑斑病和炭疽病中的应用 (Application of streptomyces diastatochromogenes metabolite in preventing and treating thunberg dwarf lilyturf black spot and anthracnose ) 是由 申屠旭萍 宋阳 曹瑱艳 俞晓平 许益鹏 刘光富 于 2020-12-14 设计创作，主要内容包括：本发明公开了一种淀粉酶产色链霉菌代谢产物在防治浙麦冬黑斑病和炭疽病中的应用,涉及微生物技术领域。本发明所涉菌株的分类命名为淀粉酶产色链霉菌(Streptomyces diastatochromogenes),编号为tz3-1,保藏日期2019年12月3日,保藏登记号CGMCC No.19069。本发明最显著的特征是该菌株液体发酵代谢产物能同时抑制浙麦冬炭疽病病原菌Colletotrichum liriopes和黑斑病病原菌Alternaria alternata菌丝的生长。田间试验表明该菌株代谢产物在防治浙麦冬黑斑病和炭疽病上作用比常规化学农药略优。(The invention discloses application of a streptomyces diastatochromogenes metabolite in preventing and treating thunberg dwarf lilyturf black spot and anthracnose, and relates to the technical field of microorganisms. The classification name of the related strain is Streptomyces diastatochromogenes (Streptomyces diastochromogenes), the number is tz3-1, the preservation date is 2019, 12 months and 3 days, and the preservation registration number is CGMCC No. 19069. The most remarkable characteristic of the invention is that the liquid fermentation metabolite of the strain can simultaneously inhibit the growth of pathogenic bacteria Colletotrichum liriopes and Alternaria alternata hyphae of the anthracnose of Thunberg dwarf lilyturf. The field test shows that the metabolite of the strain has slightly better effect on preventing and treating the black spot and the anthracnose of the Zhejiang dwarf lilyturf tuber than the conventional chemical pesticide.)

1. Application of Streptomyces diastatochromogenes metabolite in preventing and treating thunberg dwarf lilyturf black spot and anthracnose; the Streptomyces diastatochromogenes (Streptomyces diastochromogenes) is numbered tz3-1, is preserved in the China general microbiological culture Collection center, has the preservation address of No. 3 Xilu-Beijing West district of Chaoyang area, the preservation date of No. 3 is 12 months in 2019, and the preservation registration number is CGMCC No. 19069.

2. The use according to claim 1, wherein the metabolite of Streptomyces diastatochromogenes is directly used as an agent for preventing and treating black spot and anthracnose of Thunberg Ophiopogon japonicus.

3. The use according to claim 1, wherein the metabolite of Streptomyces diastatochromogenes is used as a component of an agent for preventing and treating black spot and anthracnose of Thunberg Ophiopogon japonicus.

4. Use according to claim 1, characterized in that the metabolite of Streptomyces diastochromogenes is produced by:

1) the streptomyces diastatochromogenes tz3-1 strain is activated and cultured under the condition of 28 +/-1 ℃;

2) sterilizing the fermentation culture solution, cooling, inoculating streptomyces diastatochromogenes tz3-1 under the aseptic condition, performing fermentation culture for 5 days at the temperature of 28 +/-1 ℃, centrifuging the fermentation liquor, and taking the supernatant as a metabolite of the streptomyces diastatochromogenes.

5. The use according to claim 4, wherein the fermentation broth comprises: each 1000mL of the starch concentrate contains 20g of soluble starch, CaCO₃ 5g，KH₂PO4 3g，NH₄Cl 3g，FeCl₂1g, 43g of soybean meal, 4g of yeast extract, 6g of malt extract and the balance of water.

6. Use according to claim 4, wherein the supernatant obtained by centrifugation is filtered through a 0.22 μm membrane as a metabolite of Streptomyces diastochromogenes.

Technical Field

The invention relates to the technical field of microorganisms, and in particular relates to application of a streptomyces diastatochromogenes metabolite in preventing and treating thunberg dwarf lilyturf black spot and anthracnose.

Background

Radix Ophiopogonis (Ophiopogon japonicus) is a plant of Ophiopogon genus of Liliaceae family, and its dry root tuber is used as a medicine clinically. The active ingredients of the radix ophiopogonis are saponin, flavone, amino acid, polysaccharide and the like, and the radix ophiopogonis is considered by traditional medicine to have the effects of nourishing yin, promoting the secretion of saliva, moistening lung, relieving cough and the like. Modern pharmacology finds that the radix ophiopogonis extract can diminish inflammation, the radix ophiopogonis polysaccharide has the effects of resisting thrombus, reducing blood sugar and the like, and the radix ophiopogonis saponin has the effects of relieving cough, resisting oxidation, improving liver and lung and the like. The main cultivated fields are Zhejiang and Sichuan, which are divided into Zhejiang and Sichuan ophiopogon root, and Zhejiang ophiopogon root is traditionally regarded as the best quality. Nowadays, the research and development of radix ophiopogonis medicines are more and more, various dosage forms are endlessly developed, according to the report of the development of the traditional Chinese medicine industry in Zhejiang province, the planting area of Zhejiang radix ophiopogonis reaches 3933 mu in 2015 years, and the annual sales of developed derivative Chinese patent medicine preparations such as Shenmai injection, Shengmai capsule and other products reaches more than 20 hundred million yuan, so that huge economic benefits are brought.

However, the rapid development of the Zhejiang dwarf lilyturf tuber industry also brings problems, such as non-standard cultivation technology, improper pesticide use and the like, which seriously affect the yield and quality of the Zhejiang dwarf lilyturf tuber and limit the sustainable development of the Zhejiang dwarf lilyturf tuber. Common diseases on ophiopogon root are black spot and anthracnose. According to investigation, chemical prevention and control are still the first choice in Zhe dwarf lilyturf planting areas, but the pesticide residues of finished medicinal materials exceed the standard, pathogenic bacteria generate drug resistance, the planting environment is polluted and the like. Therefore, the biological pesticide with good environmental compatibility is a better choice for preventing and treating the plant diseases of the traditional Chinese medicinal materials. Streptomyces diastochromogenes tz3-1 is a biocontrol bacterium obtained by ribosome engineering breeding in the laboratory, and metabolites of the biocontrol bacterium comprise nucleoside antibiotics and polyene macrolide antibiotics, and have strong inhibition effect on the hypha growth of various pathogenic fungi. According to the invention, the inhibition effect of the metabolite of the strain on the black spot disease and anthracnose pathogenic fungi of the Zhejiang ophiopogon root is researched by a hypha growth rate method, the field control effect of the metabolite of the strain is further determined, the effect of the metabolite of the strain on controlling the black spot disease and anthracnose of the Zhejiang ophiopogon root is found to be slightly better than that of a conventional chemical pesticide, and a new means is provided for biological control of the fungal diseases of the Zhejiang ophiopogon root. Meanwhile, the invention also provides an excellent starting strain for further development of microbial pesticides.

Disclosure of Invention

The invention aims to provide application of a biocontrol strain Streptomyces diastatochromogenes (Streptomyces diastatochromogenes) metabolite in preventing and treating thunberg dwarf lilyturf black spot and anthracnose aiming at the defects of insecurity of chemical pesticides and few varieties of microbial pesticides.

The purpose of the invention is realized by the following technical scheme:

the invention firstly discloses application of a Streptomyces diastatochromogenes metabolite in preventing and treating thunberg dwarf lilyturf black spot and anthracnose; the Streptomyces diastatochromogenes (Streptomyces diastochromogenes) is numbered tz3-1, is preserved in the China general microbiological culture Collection center, has the preservation address of No. 3 Xilu-Beijing West district of Chaoyang area, the preservation date of No. 3 is 12 months in 2019, and the preservation registration number is CGMCC No. 19069.

According to one embodiment of the invention, the metabolite of Streptomyces diastatochromogenes is directly used as an agent for preventing and treating black spot and anthracnose of Thunberg dwarf lilyturf.

According to one embodiment of the invention, the metabolite of Streptomyces diastatochromogenes is used as a component of the medicament for preventing and treating the black spot and anthracnose of Thunberg dwarf lilyturf. The reagent can also comprise other active components which are beneficial to controlling the Hangzhou white chrysanthemum leaf blight and black spot disease or optional auxiliary components, wherein the auxiliary components refer to acceptable auxiliary materials, and are typical but not limited, such as a stabilizer and a diluent.

Preferably, the metabolite of Streptomyces diastatochromogenes is prepared by the following steps:

1) the streptomyces diastatochromogenes tz3-1 strain is activated and cultured under the condition of 28 +/-1 ℃;

2) sterilizing the fermentation culture solution, cooling, inoculating streptomyces diastatochromogenes tz3-1 under the aseptic condition, performing fermentation culture at the temperature of 28 +/-1 ℃, centrifuging the fermentation liquor, and taking the supernatant as a metabolite of the streptomyces diastatochromogenes.

Preferably, the fermentation culture solution comprises the following components: each 1000mL of the starch concentrate contains 20g of soluble starch, CaCO₃5g，KH₂PO4 3g，NH₄Cl 3g，FeCl₂1g, 43g of soybean meal, 4g of yeast extract, 6g of malt extract and the balance of water.

Preferably, the supernatant obtained by centrifugation is filtered through a 0.22 μm membrane and used as a metabolite of Streptomyces diastochromogenes.

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

1) black spot and anthracnose are common diseases on radix ophiopogonis; the invention discloses a method for preventing and treating thunberg dwarf lilyturf tuber black spot and anthracnose by using a metabolite of streptomyces diastatochromogenes tz3-1 strain, which is disclosed by the invention, and is characterized in that a biological source reagent capable of effectively preventing and treating two diseases simultaneously is lacked in the prior art; compared with various problems caused by chemical control, the invention is a biological source reagent with good environmental compatibility and provides a new means for biological control of the fungal diseases of the Zhe ophiopogon japonicus.

2) The results of field control effect measurement experiments show that 100% tz3-1 metabolite has good field control effects on both ophiopogon japonicus black spot disease and anthracnose, and the correction control effects can respectively reach 74.08% and 84.37%. The field control effect of 50% tz3-1 and 50% carbendazim on the ophiopogon japonicus black spot is not different significantly. Therefore, compared with the conventional chemical pesticide, the metabolite of the streptomyces diastatochromogenes tz3-1 strain provided by the invention has better effect on preventing and treating the leaf blight and the black spot of the thunberg dwarf lilyturf tuber.

Detailed Description

Example 1: (liquid fermentation of Streptomyces diastatochromogenes tz 3-1)

Streptomyces diastochromogenes tz3-1 is obtained by ribosome engineering breeding of Streptomyces diastochromogenes D, and is specifically disclosed in the patent application (application number: 2019114179541).

The fermentation culture solution contains 20g of soluble starch and CaCO per 1000mL₃ 5g，KH₂PO4 3g，NH₄Cl 3g，FeCl₂1g, 43g of soybean meal, 4g of yeast extract, 6g of malt extract,the balance of water; bottling 60mL of fermentation culture solution in each 300mL triangular flask, sterilizing at 121 deg.C for 20min, cooling to 50 deg.C, inoculating Streptomyces platinum-loop amylase chromogenes D or corresponding mutant strain under aseptic condition, fermenting at 28 + -1 deg.C for 5 days; the fermentation liquor is centrifuged for 10min at 5000r/min, and the supernatant is filtered by a 0.22 mu m membrane and then used for antibacterial activity determination and field control effect test.

Example 2: (determination of bacteriostatic Activity of Streptomyces diastatochromogenes tz3-1 metabolite)

Taking 1mL of fermentation supernatant after being filtered by a 0.22-micron membrane, putting the fermentation supernatant into a sterile culture dish, quickly and uniformly mixing the fermentation supernatant with 9mL of PDA culture medium cooled to 50 ℃, respectively putting 1 test bacterium cake with the diameter of 5mm on the plane of the culture medium after cooling, inoculating the bacterium cake into the center of the culture dish (the diameter of the culture dish is 9cm), putting the culture dish into an incubator, culturing for 36h at 28 ℃, and repeating for 3 times by taking sterile water treatment as a reference; the colony diameter was measured by the cross method, and the inhibition rate was calculated by the following formula: the hyphal growth inhibition ratio (%) [ (control colony diameter-5) - (treated colony diameter-5) ]/(control colony diameter-5) × 100%.

The measurement result shows that: streptomyces diastochromogenes tz3-1 metabolite has good inhibition effect on the growth of hyphae of 2 pathogenic fungi to be tested, and the inhibition rate is more than 98%.

TABLE 1 inhibitory Effect of Streptomyces diastatochromogenes tz3-1 metabolite on pathogenic fungi of fungal diseases of Zhejiang ophiopogon root

The inhibitory effect (%)^aThe column data is the inhibition effect data of the metabolite obtained by culturing the medium and the culture method disclosed in the application number CN201911417954.1 on the fungus pathogenic fungi of the Thunberg dwarf lilyturf tuber (the metabolite is the supernatant obtained by centrifuging the fermentation broth and is not treated by the step of separating tetramycin Z)High.

Example 3: (determination of field prevention of metabolite of Streptomyces diastochromogenes tz 3-1)

100 percent, 50 percent of metabolite of streptomyces diastatochromogenes tz3-1 and 50 percent of carbendazim are selected as test reagents for field control effect determination, clear water is used as negative control, and the negative control treated by the clear water between each treated field is used as a field spacing. And (5) investigating the disease condition in 7d, counting the disease index, and calculating the correction control effect. The disease is classified into 4 grades according to the severity of the disease: grade 0, healthy seedlings; grade 1, at least 25% of the leaves die; grade 2, 26% -50% of leaves die; grade 3, 51% -75% of leaves wither; grade 4, 76% -100% of the leaves die. The disease index [ Σ (number of diseased plants at each stage × representative value at each stage)/(total number of investigated plants × representative value at the highest level of disease) ] × 100; control effect (%) corrected by 100 × (1- (post-treatment agent disease means × pre-control agent disease means)/(pre-treatment agent disease means × post-control agent disease means)).

The results of the experiment (table 2) show that: the 100% tz3-1 metabolite has good field control effects on the black spot and anthracnose of radix ophiopogonis, and the correction control effects can respectively reach 74.08% and 84.37%. The field control effect of 50% tz3-1 and 50% carbendazim on the ophiopogon japonicus black spot is not different significantly.

TABLE 2 prevention and treatment effects of Streptomyces diastatochromogenes tz3-1 metabolite on fungus diseases of Zhejiang ophiopogon root

Note: 100% tz3-1 refers to direct fermentation to obtain supernatant filtered through 0.22 μm membrane;

50% tz3-1 refers to a solution of 100% tz3-1 supernatant diluted with sterile water in a volume ratio of 1: 1;

after the control effect numerical value is corrected, different lower case letters indicate that the control effect is remarkably different among different biocontrol agents (P is less than 0.05).