阅读说明：本技术 淀粉酶产色链霉菌代谢产物在防治铁皮石斛病害中的应用 (Application of streptomyces diastatochromogenes metabolite in preventing and treating dendrobium officinale diseases ) 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铁皮石斛病害中的应用,涉及微生物技术领域,本发明所述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铁皮石斛炭疽病病原菌(Colletotrichum gloeosporioides)、根腐病病原菌(Fusarium chlamydosporum)、梢枯病病原菌(Alternaria alternata)、叶斑病病原菌(Cladosporium cladosporioides)和白绢病病原菌(Sclerotium delphinii)菌丝的生长。田间试验表明该菌株代谢产物在防治铁皮石斛梢枯病上作用比常规化学农药略优。(The invention discloses an application of a metabolite of Streptomyces diastatochromogenes in preventing and treating diseases of dendrobium officinale, and relates to the technical field of microorganisms, wherein the strain is classified and named as Streptomyces diastatochromogenes (Streptomyces diastchromogenes) with the number of tz3-1, the preservation date of 2019, 12 months and 3 days, and the preservation registration number of 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 the hyphae of dendrobium candidum anthracnose pathogenic bacteria (Colletotrichum gloeosporioides), root rot pathogenic bacteria (Fusarium chlamydosporum), tip blight pathogenic bacteria (Alternaria alternata), leaf spot pathogenic bacteria (Cladosporium cladosporioides) and southern blight pathogenic bacteria (Sclerotium delphinii). The field test shows that the metabolite of the strain has slightly better effect on preventing and treating the dendrobium candidum dieback than the conventional chemical pesticide.)

1. An application of Streptomyces diastatochromogenes metabolite in preventing and treating diseases of Dendrobium officinale is provided; 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, characterized in that the metabolite of Streptomyces diastatochromogenes is used directly as a medicament for controlling diseases of dendrobium officinale.

3. The use according to claim 1, characterized in that the metabolite of Streptomyces diastatochromogenes (Streptomyces diastatochromogens) is used as a constituent in a medicament for controlling diseases of dendrobium officinale.

4. The use of claim 1, wherein the disease is a plurality of anthracnose, dieback, leaf spot, root rot, southern blight.

5. The use of claim 1, wherein the disease is a dieback disease.

6. 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.

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

8. Use according to claim 6, 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 particularly relates to application of a streptomyces diastatochromogenes metabolite in preventing and treating dendrobium officinale diseases.

Background

Dendrobium officinale (Dendrobium officinale) is a perennial herb of the genus Dendrobium of the family Orchidaceae, the main medicinal part is a fresh or dried stem, and is a very precious Chinese medicinal material. The traditional medicine considers that the dendrobium officinale has the functions of reinforcing stomach, promoting fluid production, nourishing yin, clearing heat and improving immunity. Modern pharmacology finds that the dendrobium officinale has good regulating effects on a digestive system, an immune system, diabetic angiopathy and the like, and has the effects of resisting oxidation, ageing, fatigue and tumors, protecting liver and the like. The main active substances are reported to be sesquiterpenoids, mucoids, sterols, phenolic compounds, and the like. In China, the dendrobium officinale is mainly produced in regions with the elevation of more than 1600m in Yunnan province and Zhejiang province, but due to various factors such as strict requirements on the growth environment, slow growth speed, excessive collection and the like, the dendrobium officinale is listed as endangered medicinal plants since 1987. In recent years, with the development of scientific technology, breeding technology, tissue culture, artificial cultivation technology and the like, the method is widely applied to the planting of the dendrobium officinale, the yield and the quality of the dendrobium officinale are greatly improved, the current planting area of the dendrobium officinale in Zhejiang is counted to be 3.5 mu per thousand, the yield exceeds 40 billion yuan, and the dendrobium officinale accounts for more than 70% of the whole country.

However, the rapid development of the dendrobium officinale 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 dendrobium officinale and limit the sustainable development of the dendrobium officinale. According to statistics, the diseases of the dendrobium officinale can be dozens of diseases, and the common diseases comprise black spot, anthracnose, epidemic disease, southern blight, root rot, gray mold and the like. Investigation finds that chemical prevention and control are still the first choice in the planting areas of the traditional Chinese medicinal materials, but the chemical prevention and control also causes various problems of the finished medicinal materials, such as standard exceeding of pesticide residues, generation of drug resistance of pathogenic bacteria, pollution to the planting environment 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 pathogenic fungi of the dendrobium candidum pin blight, pathogenic bacteria of anthracnose and the like is researched by a hypha growth rate method, the field prevention effect is further measured, the effect of the metabolite of the strain on preventing and treating the dendrobium candidum pin blight is slightly better than that of a conventional chemical pesticide, the metabolite of the strain also shows good prevention and treatment effects on root rot and southern blight, and a new means is provided for biological prevention and treatment of dendrobium candidum fungal diseases. 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 diastochromogenes) metabolite in preventing and treating diseases of dendrobium officinale by 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 diseases of dendrobium officinale; 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.

The streptomyces diastatochromogenes tz3-1 can obtain a metabolite with prevention and treatment effects on part of diseases of dendrobium officinale by liquid fermentation.

In a preferred embodiment, the metabolite of the streptomyces diastatochromogenes is directly used as a medicament for controlling dendrobium officinale diseases.

In another preferred embodiment, the metabolite of streptomyces diastatochromogenes is used as a component in an agent for controlling dendrobium candidum diseases, and the agent can also comprise other active components beneficial to controlling dendrobium candidum diels or optional auxiliary components, wherein the auxiliary components refer to acceptable auxiliary materials, typically but not limited to, such as a stabilizer and a diluent.

The diseases of the dendrobium officinale are various diseases of anthracnose, dieback, leaf spot, root rot and southern blight, and the metabolic product of the streptomyces diastatochromogenes can simultaneously generate control effects on various diseases.

Preferably, the disease is the pin blight, and the field control effect shows that the metabolite of the strain has better effect on preventing and treating the pin blight of the dendrobium officinale than the conventional chemical pesticide.

Preferably, the metabolite of the amylase streptomyces chromogenes can be 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 for 5 days at the temperature of 28 +/-1 ℃, centrifuging the fermentation liquor, and taking the supernatant as a metabolite of the streptomyces diastatochromogenes.

In a preferred embodiment, the fermentation broth consists of: each 1000mL of the starch concentrate contains 20g of soluble starch, CaCO₃ 5g，KH₂PO4 3g，NH₄Cl 3g，FeCl₂1g of soybean meal, 43g of soybean meal and the balance of water.

In a preferred embodiment, the supernatant obtained by centrifugation is filtered through a 0.22 μm membrane as a metabolite of Streptomyces diastochromogenes.

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

1) the invention discovers that the metabolite of the streptomyces diastatochromogenes tz3-1 strain can be used for preventing and treating diseases such as anthracnose, dieback, leaf spot, root rot, southern blight and the like of dendrobium officinale; compared with the chemical prevention and control of the pesticide residues of the finished medicinal materials exceeding the standard, pathogenic bacteria generating drug resistance, the pollution to the planting environment and other problems, the invention belongs to the biological pesticide prevention and control and has good environmental compatibility.

2) The field control effect test result shows that the control effect of 50% tz3-1 on the dendrobium candidum dieback is not obviously different from that of 100% tz3-1, but the control effect is superior to that of positive control 50% carbendazim in the field control effect test result of southern blight, and the control effect of 100% tz3-1 metabolite is equivalent to that of 50% carbendazim. Therefore, the Streptomyces diastochromogenes tz3-1 metabolite is better than conventional chemical pesticides in the effect of preventing and treating the dendrobium candidum dieback, can avoid the problem caused by long-term application of chemical drugs, and is an extremely efficient means for preventing and treating the dendrobium candidum dieback.

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 and the balance of water; bottling 60mL of fermentation culture solution in each 300mL triangular flask, sterilizing at 121 ℃ for 20min after preparation, selecting a platinum-ring amylase chromogen D or a corresponding mutant strain to inoculate when cooling to 50 ℃ under the aseptic condition, and carrying out fermentation culture at 28 +/-1 ℃ for 5D; 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 hypha growth of 5 pathogenic fungi to be tested.

TABLE 1 inhibitory Effect of Streptomyces diastatochromogenes tz3-1 metabolite on pathogenic fungi of Dendrobium officinale fungal diseases

The inhibitory effect (%)^aThe column data is the inhibition effect data of the metabolite obtained by culturing the culture medium and the culture method in application number CN201911417954.1 on the pathogenic fungi of the dendrobium candidum fungal disease (the metabolite is the supernatant obtained by centrifuging the fermentation liquor and is not treated by the step of separating tetramycin Z).

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 control effect of 50% tz3-1 on the dendrobium officinale dieback is not obviously different from that of 100% tz3-1, but the control effect is better than that of positive control 50% carbendazim; the best effect medicament for the anthracnose of the dendrobium officinale is carbendazim, the prevention and treatment effect is 87.57%, and the prevention and treatment effect is obviously higher than that of a tz3-1 metabolite; in addition, in the field control experiment result of southern blight, the control effect of 100% tz3-1 metabolite is equivalent to that of 50% carbendazim.

TABLE 2 prevention and treatment of fungal diseases of Dendrobium officinale by Streptomyces diastatochromogenes tz3-1 metabolite

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).