CN113303341A - Application of streptomyces malaysiae F913 - Google Patents
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Abstract
The invention provides application of streptomyces malaysiae F913, and particularly relates to the field of disease control. The invention provides application of streptomyces malaysiae F913 in preventing and treating animal and plant pathogenic fungi, and the streptomyces malaysiae F913 provided by the invention can avoid the occurrence of the conditions of environmental pollution, fruit pesticide residue, induced pathogenic bacteria generating drug resistance and the like, and has excellent prevention effect, time saving and labor saving.
Description
Technical Field
The invention relates to the field of disease control, in particular to application of streptomyces malaysiae F913.
Background
Currently, the prevention and control means for pathogenic fungi of animals and plants, such as isaria farinosa (Isariafarinosa), Verticillium dahliae (Verticillium dahliae), Colletotrichum cucurbitacearum (Colletotrichum lagenarium), Rhizoctonia solani (Rhizoctonia solani), Fusarium solani (Fusarium solani), and the like, are mainly chemical control, and are supplemented with various prevention and control means such as physical control, agronomic control, resistance breeding, and the like. Although chemical pesticides can effectively prevent and control the occurrence of pathogenic fungi in a short period, a series of defects such as environmental pollution, fruit pesticide residue, pathogenic bacteria drug resistance and the like are easily caused by long-term use of the chemical pesticides; the physical and agricultural control means have the problems of time and labor consumption, limited control effect and the like; and the improvement of many animal and plant pathogenic fungi resistant varieties is difficult to achieve breakthrough development in a short time. In recent years, with the enhancement of environmental awareness and the increasing emphasis on the demand for green foods, biological control methods for animal and plant diseases by using microorganisms have been receiving more and more attention.
Disclosure of Invention
In order to solve the problems, the invention provides an application of streptomyces malaysiensis F913. The strain provided by the invention can avoid the conditions of environmental pollution, fruit pesticide residue and drug resistance generation of induced pathogenic bacteria, and has excellent prevention effect, and time and labor are saved.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides application of streptomyces malaysiae F913 in preventing and treating animal and plant pathogenic fungi.
Preferably, the animal pathogenic fungi of the animal and plant pathogenic fungi include isaria farinosa.
Preferably, the plant pathogenic fungi of the animal and plant pathogenic fungi comprise verticillium dahliae, anthracnose of cucumber, rhizoctonia solani, trichoderma viride, fusarium solani and phytophthora nicotianae.
The invention provides a microbial inoculum for preventing and treating animal and plant pathogenic fungi, and the effective component of the microbial inoculum comprises streptomyces malaysiae F913.
Preferably, the animal pathogenic fungi of the animal and plant pathogenic fungi include isaria farinosa; the plant pathogenic fungi include Verticillium dahliae, Colletotrichum cucumerinum, Rhizoctonia solani, Trichoderma viride, Fusarium solani and Phytophthora nicotianae.
Has the advantages that: the invention provides application of streptomyces malaysiae F913 in preventing and treating animal and plant pathogenic fungi. The strain provided by the invention can avoid the conditions of environmental pollution, fruit pesticide residue and drug resistance generation of induced pathogenic bacteria, and has excellent prevention effect, and time and labor are saved. The streptomyces malaysiae F913 strain can effectively prevent and control animal and plant pathogenic fungi.
Biological preservation Instructions
The Streptomyces malaysiensis (Streptomyces malaysiensis) F913 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of No. 1 Siro-Chen-Xilu in the Chaoyang district of Beijing, the preservation date is 10 and 22 days in 2020 and the preservation number is CGMCC No. 20941.
Drawings
FIG. 1 is a photograph showing morphological characteristics of Streptomyces malaysiae F913 in example 1;
FIG. 2 is a phylogenetic tree of Streptomyces malaysiae F913 of example 3;
FIG. 3 is a graph showing the effect of different media on the bacteriostatic effect of Streptomyces malaysiae F913 fermentation broth in example 4; the method comprises the following steps of A, a step of obtaining a fermentation broth of Streptomyces malaysiensis F913 by using a culture medium I, a step of obtaining a fermentation broth of Streptomyces malaysiensis F913 by using a culture medium II, a step of obtaining a fermentation broth of Streptomyces malaysiensis F913 by using an ISP (ISP) culture medium I, a step of obtaining an influence graph of the effect of the fermentation broth of Streptomyces malaysiensis F913 by using an ISP culture medium II, a step of obtaining an influence graph of the effect of the fermentation broth of Streptomyces malaysiensis F913 by using a soybean meal culture medium II, a step of obtaining a column graph of the effect of the fermentation broth of Streptomyces malaysiensis F913 by using different culture media F, a step of obtaining a cell growth condition in different culture media G, a step of obtaining the culture medium I by using the culture medium I, a step of obtaining the culture medium II, a step of obtaining the SP culture medium II, a step of obtaining the ISP culture medium I, a step of obtaining the culture medium II, a step of obtaining the ISP culture medium 1, a step of obtaining the ISP culture medium IV, and a step of obtaining the step I;
FIG. 4 is a graph showing the effect of different fermentation times, fermentation temperatures and initial pH values of the medium on the bacteriostatic effect of the fermentation broth of Streptomyces malaysiae F913 in example 5; wherein a is an influence graph of different fermentation times on the bacteriostatic effect of the Streptomyces malaysiae F913 fermentation broth, b is an influence graph of different fermentation temperatures on the bacteriostatic effect of the Streptomyces malaysiae F913 fermentation broth, and c is an influence graph of different initial pH values of a culture medium on the bacteriostatic effect of the Streptomyces malaysiae F913 fermentation broth;
FIG. 5 shows the diameter of the Streptomyces malaysiae F913 in inhibiting phytopathogens in example 6;
FIG. 6 is a graph showing the antagonistic effect of Streptomyces malaysiae F913 on animal pathogens in example 7.
Detailed Description
The invention provides application of streptomyces malaysiae F913 in preventing and treating animal and plant pathogenic fungi. In the present invention, the animal pathogenic fungi among the animal and plant pathogenic fungi preferably include isaria farinosa; the plant pathogenic fungi of the animal and plant pathogenic fungi preferably include Verticillium dahliae (Verticillium dahliae), Colletotrichum cucumerinum (Colletotrichum lagenarium), Rhizoctonia solani (Rhizoctonia solani), Trichoderma viride (Trichoderma viride), Fusarium solani (Fusarium solani), and Phytophthora nicotianae (Phytophthora nicotianae).
The streptomyces malaysiae F913 provided by the invention can avoid the situations of environmental pollution, fruit pesticide residue and drug resistance generation of induced pathogenic bacteria, and has excellent prevention effect, and time and labor conservation.
The invention provides a microbial inoculum for preventing and treating animal and plant pathogenic fungi, and the effective component of the microbial inoculum comprises streptomyces malaysiae F913. In the present invention, the animal pathogenic fungi among the animal and plant pathogenic fungi preferably include isaria farinosa; the plant pathogenic fungi of the animal and plant pathogenic fungi preferably include Verticillium dahliae, Colletotrichum cucumerinum, Rhizoctonia solani, Trichoderma viride, Fusarium solani and Phytophthora nicotianae.
In order to further illustrate the present invention, the following detailed description of the application of Streptomyces malaysiae F913 in controlling phytopathogenic fungi provided by the present invention is given in connection with the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1
10g of the collected southwest university landscape pond soil sample is weighed and placed into a triangular flask containing 90mL of sterile water (with a small amount of glass beads), and the mixture is fully shaken for 30 min. 1mL of the soil suspension was pipetted into 9mL of sterile physiological saline and thoroughly shaken. Then a pipette is used to aspirate 1mL of the soil suspension from the test tube and mix it with another sterile saline solution containing 9mL of the soil suspension, and the soil suspension is made to have a concentration of 10 in the same manner-1、10-2(i.e., 1mL of 10-1Mixing the dilution into a test tube containing 9mL of sterile physiological saline), 10-3(i.e., 1mL of 10-2Mixing the dilution into a test tube containing 9mL of sterile physiological saline), 10-4(i.e., 1mL of 10-3Mixing the diluent into a test tube containing 9mL of sterile saline), 10-5(i.e., 1mL of 10-4Mixing the dilution into a test tube containing 9mL of sterile physiological saline), 10-6(i.e., 1mL of 10-5The dilution was mixed into a test tube containing 9mL of sterile physiological saline). Pipette 200. mu.L of 10-4、10-5、10-6The soil dilutions in three concentrations were spread on solid culture medium (soluble starch 20g, KNO) synthesized in Gao's synthetic No. one31g、NaCl 0.5g、K2HPO40.5g、MgSO4·7H20.5g of O and FeSO4·7H2O0.01 g, distilled water to 1L, agar 20g, pH adjusted to 7.4), 3 replicates per gradient setup. The solid plate was placed in a 25 ℃ incubator and continuously incubated for 1 week, and observed day by day. After colonies of actinomycetes grew on the plate, single colonies were isolated by streaking and inoculated on a soybean powder solid medium (20 g of soybean powder and 20g of mannitol, ddH)2O to 1L, agar 20g, pH 7.0), culturing at 30 ℃ for 7d, collecting spores to obtain Streptomyces malaysiae F913 bacterial suspension, and preserving with 20% glycerol; stored in a refrigerator at-80 ℃.
The morphological characteristics of the streptomyces malaysiae F913 are measured.
Morphological characteristics: the colony is white in the initial stage, gradually turns to grey with the formation of spores, the final color of the colony is grey, no water-soluble pigment is produced, the aerial hyphae of the streptomyces malaysiensis F913 are more developed, and the spore silk is spiral, as shown in figure 1.
Example 2
The physiological and biochemical detection is carried out on the streptomyces malaysiae F913, and the detection result is shown in table 1.
TABLE 1 physiological and biochemical indices of Streptomyces malaysiae F913
Note: "+" indicates that the test reaction was positive; "-" indicates that the test reaction was negative.
The results are shown in Table 1, in which Streptomyces malaysiae F913L-arabinose was positive, D-xylose was positive, D-fructose was positive, L-rhamnose was positive, D-mannitol was positive, L-inositol was positive, sucrose was positive, raffinose was positive, gelatin liquefaction was negative, milk coagulation and peptonization was positive, starch hydrolysis was positive, cellulose hydrolysis was negative, H-inositol was positive, and2s production is negative, and the strain can also secrete amylase to hydrolyze starch and solidify milk, but can not liquefy gelatin and can not decompose cellulose.
Example 3
The sequence 1391bp of the gene is obtained by amplifying and sequencing the 16SrDNA sequence of the streptomyces malaysiensis F913, and the sequence is submitted to a GenBank database, wherein the accession number is KP 338102. The sequence is compared with the sequence in GenBank on line, the result shows that the homology of the sequence and the 16SrDNA gene sequence of a plurality of strains of Streptomyces malaysiensis is up to 100 percent, and the phylogenetic result based on 16S rDNA shows that the Streptomyces malaysiensis F913 and the Streptomyces malaysiensis NBRC 16446 strain with the accession number of NR041410 are in the same minimum branch in a phylogenetic tree, and the result is shown in figure 2.
Example 4
Influence of different culture media on the bacteriostatic effect of the Streptomyces malaysiae F913 fermentation broth.
Preparation of a streptomyces malaysiensis F913 fermentation seed liquid:
inoculating Streptomyces malaysiae F913 to MS solid culture medium, culturing at 30 deg.C for 7d, collecting spores with 0.05% Tween-20 to obtain spore suspension; the spore suspension was transferred to a 1L Erlenmeyer flask containing 100mL of Gao's synthetic No. one liquid medium and cultured at 30 ℃ and 200rpm for 3 days.
The influence of the fermentation medium on the bacteriostatic effect of the Streptomyces malaysiensis F913 fermentation broth is as follows:
the prepared seed solutions were inoculated into Gao synthetic No. one medium (soluble starch 20g, KNO) in an amount of 1% respectively31g、NaCl 0.5g、K2HPO40.5g、MgSO4·7H20.5g of O and FeSO4·7H20.01g of O, distilled water with constant volume of 1L, pH adjusted to 7.4), SP medium (30 g of mannitol, 10g of potato starch, 5g of neutral soybean peptone, 8g of yeast extract, distilled water with constant volume of 1L, pH adjusted to 6.0), ISP medium No. 1 (5 g of casein peptone, 3g of yeast extract, 15g of agar, ddH)2O to volume of 1L, pH 7.0), ISP culture medium No.2 (purchased from chemical Co., Ltd., product No. HW-HBHB8746 of Beijing Huawei Rui), and semen glycines powder culture medium (semen glycines powder 20g, mannitol 20g, ddH2O is constant volume to 1L, pH is 7.0), liquid loading amount is 200mL (1L conical flask), fermentation is carried out for 7d at 30 ℃ and 200 rpm;
each group had 3 replicates; and (3) measuring the effect of antagonistic entomopathogenic fungus isaria of the fermented supernatant by using a bacteriostatic circle method. The results are shown in FIG. 3 and Table 2.
TABLE 2 diameter of zone of inhibition of Streptomyces malaysiae F913 by different fermentation media
As shown in FIG. 3 and Table 2, the antagonistic action was exhibited by the produced fermentation broths, but the antagonistic effect was different in the fermentation broths obtained by the fermentation in different media. When the fermentation culture medium is SP culture medium, ISP culture medium No. 1 culture medium and ISP culture medium No.2 culture medium, the antagonistic effect of the fermentation liquid is the best, the second time of synthesizing the first culture medium by Gao's method, the worst of the soybean powder culture medium; however, when the SP culture medium is used for fermentation, more sediment substances are generated, and the later fermentation liquid treatment is not facilitated, so that the ISP culture medium No.2 is finally determined as the optimal culture medium.
Example 5
The influence of different fermentation time, fermentation temperature and initial pH value of the culture medium on the bacteriostasis effect of the fermentation liquor of the streptomyces malaysiensis F913.
Influence of fermentation time on bacteriostatic effect of Streptomyces malaysiae F913 fermentation broth
The seed solution prepared in example 4 was inoculated into ISP medium No.2 at an inoculum size of 1%, the liquid loading volume was 200mL (1L Erlenmeyer flask), and fermentation was carried out at 30 ℃ and 200rpm for 3d, 5d, 7d, and 9d, respectively; each group had 3 replicates; and (3) determining the antibacterial effect of the fermentation supernatant by an antibacterial ring method. The results are shown in FIG. 4a and Table 3.
TABLE 3 diameter of zone of inhibition for Streptomyces malaysiae F913 for different fermentation times
Influence of fermentation temperature on bacteriostatic effect of Streptomyces malaysiae F913 fermentation broth
Respectively inoculating the prepared seed solutions to ISP culture medium No.2 according to the inoculation amount of 1%, wherein the liquid loading amount is 200mL (1L conical flask), respectively placing at 20 ℃, 25 ℃, 30 ℃, 35 ℃, 200rpm, and fermenting for 7 d; each group had 3 replicates; and (3) determining the antibacterial effect of the fermentation supernatant by an antibacterial ring method. The results are shown in FIG. 4b and Table 4.
TABLE 4 different fermentation temperatures for the zone of inhibition of Streptomyces malaysiae F913
Influence of initial pH value of culture medium on bacteriostatic effect of Streptomyces malaysiae F913 fermentation broth
Adjusting the initial pH value of the ISP culture medium No.2 to be 5, 6, 7, 8 and 9 respectively by using 0.4mol/L of LNaOH solution or 0.4mol/L of HCl solution; then inoculating according to the inoculation amount of 1%, wherein the liquid loading amount is 200mL (1L conical flask), the temperature is 30 ℃, the rpm is 200, and the fermentation is carried out for 7 d; each group had 3 replicates; and (3) determining the antibacterial effect of the fermentation supernatant by an antibacterial ring method. The results are shown in FIG. 4c and Table 5.
TABLE 5 fermentation Medium different initial pH vs. inhibition zone diameter of Streptomyces malaysiae F913
As can be seen from FIG. 4 and tables 3 to 5: when the fermentation medium is ISP medium No.2, fermentation 7d, 9d, the antagonistic effect of the fermentation liquid is the best, and fermentation is performed 3d, 5d times, so that the optimal fermentation time is determined to be 7d (a in fig. 4). Streptomyces malaysiae F913 is fermented and cultured at 20 ℃, 25 ℃, 30 ℃ and 35 ℃, and the produced fermentation liquor has antagonistic activity, but has different bacteriostatic effects at different temperatures (b in figure 4). The bacteriostatic effect is poor after the culture at 20 ℃ and 25 ℃; the bacteriostatic effect is good and has no obvious difference after the culture at the temperature of 30 ℃ and the culture at the temperature of 35 ℃, so the selective culture temperature is 30-35 ℃. Adjusting the initial pH value of the culture medium to 5, 6, 7, 8 and 9 respectively, and measuring the antagonistic activity of the fermentation liquor by using a bacteriostatic circle method; the result shows that when the initial pH value is 5, the strain F913 hardly produces the bacteriostatic active substances, when the initial pH value of the culture medium is 6-9, the Streptomyces malaysiensis F913 can produce the bacteriostatic active substances, and when the initial pH value of the culture medium is 7, the bacteriostatic active substance producing capability is strongest, and the fermentation broth antagonistic effect is best (c in FIG. 4).
Example 6
The experimental method comprises the following steps: and detecting the antibacterial potency of the supernatant obtained by fermenting the strain F913 by adopting a hypha growth rate method.
Mixing the sterile fermentation supernatant with sterilized PDA culture medium cooled to 55 ℃ in a ratio of 1:10, namely, in the embodiment, 2ml of supernatant is mixed with 20ml of culture medium, a PDA plate without fermentation supernatant is used as a blank control, a pathogenic bacteria cake with the diameter of 5mm is inoculated, each treatment is repeated for 3 times, the culture is carried out at constant temperature of 22 ℃, the growth diameters of pathogenic bacteria of an experimental group and a control group are observed and compared day by day, wherein the pathogenic fungi comprise: the plant pathogens Verticillium dahliae (v. dahliae), cucumopsis sativus (c. lagenaria), Rhizoctonia solani (r. solani), Trichoderma viride (t. viride), fusarium solani (f. solani) and Phytophthora nicotianae (p. nicotianae).
The specific method is that a puncher is utilized to punch a 5mm fungus cake on a 5 d-growing fungus lawn and place the fungus cake on an inhibition plate, and the inhibition index is adopted to express the effect. The results are shown in Table 6 and FIG. 5.
Wherein, the formula of the inhibition index is as follows: inhibition index is 1-Da/Db (Da: colony diameter on inhibition plate. Db: colony diameter on control plate), formula i.
TABLE 6 fungal inhibition index of Streptomyces malaysiae strain F913
| Plant pathogenic bacteria | Inhibition index | Plant pathogenic bacteria | Inhibition index |
| T.viridae | 54.51 | V.dahliae | 70.35 |
| R.solani | 61.99 | C.lagenarium | 78.05 |
| F.solania | 57.2 | P.nicotianae | 53.73 |
As can be seen from Table 6 and FIG. 5, the Streptomyces malaysiae F913 provided by the present invention has a good inhibitory effect on Verticillium dahliae, Colletotrichum cucumerinum, Rhizoctonia solani, Trichoderma viride, Fusarium solani and Phytophthora nicotianae.
Example 7
In this example, two experiments were designed for detecting the antagonistic effect of streptomyces malaysiensis F913 on isaria pulcherrima in soil:
large-particle stones in the collected soil sample are removed, the soil sample is sieved and is dried in an oven at 65 ℃ overnight. After the soil is cooled, adding streptomyces malaysiensis F913 and isaria pulcherrima suspension with the addition concentration of 108cfu/g. The preparation method of the isaria pinicola suspension comprises the following steps: the suspension was prepared by adding water to the culture plate, followed by scraping surface spores with a cotton swab, and then filtering the suspension through absorbent cotton to remove the mycelia.
One group of: adding Streptomyces malaysiae F913 suspension, adding Isaria farinosa If suspension (F913-If) after one week, and adding ddH2O as a control, i.e. without addition of the suspension of Streptomyces malaysiae F913, an equivalent amount of ddH was added2O(ddH2O- -If); wherein the viable count of the streptomyces malaysiensis F913 suspension and the isaria pulcherrima suspension is 108cfu/g。
Two groups are as follows: adding isaria pulcherrima If suspension, and adding streptomyces malaysiensis F913 after one weekSuspension (If- -F913) in ddH2O as a control (If- -ddH)2O)。
The method comprises the following steps:
F913-If and ddH are counted by coating counting2O-If, If-F913 and If-ddH2The detection results of cfu of isaria switzeri in soil at 0, 5, 7, 14 and 21d are shown in table 7 and fig. 6.
TABLE 7 cfu data of Isaria farinosa in various cultivation methods and cultivation times
The results of the experiments shown in Table 7 and FIG. 6 show that, when Streptomyces malaysiensis F913 and Isaria farinosa (F913- -If) were added to the soil environment, the number of Isaria farinosa was continuously decreased as a whole and was significantly lower than that of the control group (ddH)2O-If), which proves that the addition of the streptomyces malaysiae F913 can obviously inhibit the isaria pulcherrima in the soil.
The above description shows that the streptomyces malaysiae F913 provided by the present invention can significantly inhibit verticillium dahliae, anthracnose of cucumber, rhizoctonia solani, trichoderma viride, fusarium solani, phytophthora nicotianae, and isaria farinosa, and can avoid the occurrence of environmental pollution, pesticide residue on fruits, and the occurrence of drug resistance induced by pathogenic bacteria, thereby saving time and labor.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (5)
1. Application of streptomyces malaysiae F913 in preventing and treating animal and plant pathogenic fungi.
2. The use according to claim 1, characterized in that the animal pathogenic fungi of said animal and plant pathogenic fungi comprise isaria swipes.
3. The use according to claim 1, wherein the plant pathogenic fungi of the animal and plant pathogenic fungi include verticillium dahliae, colletotrichum cucumerinum, rhizoctonia solani, trichoderma viride, fusarium solani and phytophthora nicotianae.
4. The microbial inoculum for preventing and treating animal and plant pathogenic fungi is characterized in that the effective component of the microbial inoculum comprises streptomyces malaysiae F913.
5. The microbial inoculum according to claim 4, characterized in that the animal pathogenic fungi of the animal and plant pathogenic fungi comprise Isaria farinosa; the plant pathogenic fungi include Verticillium dahliae, Colletotrichum cucumerinum, Rhizoctonia solani, Trichoderma viride, Fusarium solani and Phytophthora nicotianae.
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| CHENG, JINHUA ET AL.: "Azalomycin F complex is an antifungal substance produced by Streptomyces malaysiensis MJM1968 isolated from agricultural soil", 《JOURNAL OF THE KOREAN SOCIETY FOR APPLIED BIOLOGICAL CHEMISTRY》 * |
| PALANIYANDI, SASIKUMAR ARUNACHALAM ET AL.: "Foliar Application of Extract from an Azalomycin-Producing Streptomyces malaysiensis Strain MJM1968 Suppresses Yam Anthracnose Caused by Colletotrichum gloeosporioides", 《JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY》 * |
| 徐耀波 等: "1株抑制铜绿微囊藻生长的放线菌的分离鉴定", 《西南大学学报（自然科学版）》 * |
| 徐耀波 等: "马来西亚链霉菌F913的生物活性分析与次级代谢产物挖掘", 《2015年中国微生物学会学术年会论文集》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113999794A (en) * | 2021-11-12 | 2022-02-01 | 中国热带农业科学院热带生物技术研究所 | Streptomyces and application thereof |
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| Publication number | Publication date |
|---|---|
| CN113303341B (en) | 2021-12-10 |
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