Application of Streptomyces marshlandii strain 13-3 and its fermentation liquid in controlling corn stalk rot

Abstract

The present invention relates to an application of a wetland Streptomyces strain 13-3 in preventing and controlling corn stalk rot. The wetland Streptomyces strain 13-3 is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC No. 22830 and a deposit date of July 6, 2021. The invention has the advantages that the wetland Streptomyces strain 13-3 has an antagonistic effect on two pathogens (Fusarium graminearum and Fusarium pseudo-verticillium) that cause corn stalk rot, and its fermentation liquid can significantly inhibit the hyphae growth and spore germination of the two pathogens (Fusarium graminearum and Fusarium pseudo-verticillium) that cause corn stalk rot, and the fermentation liquid has an excellent prevention and control effect on corn stalk rot.

Description

Technical Field

[0001] The invention relates to the technical field of crop disease prevention and control, and specifically to application of a Streptomyces paludis strain 13-3 and a fermentation liquid thereof in preventing and controlling corn stalk rot. Background Art

[0002] Corn is my country's main food crop, feed crop and important industrial raw material, and occupies an important position in the national economy and agricultural production. my country is a major corn producer and importer. In 2021, my country's corn planting area increased by 5% compared with 2020, reaching 43.5 million hectares, and the output reached 272.5 million tons (https: / / www.askci.com / news / data / chanxiao / 20220119 / 1145301728429.shtml). Since 2020, China's corn imports have increased rapidly, reaching 28.35 million tons in 2021, a year-on-year increase of 222.9% (https: / / www.askci.com / news / data / maoyi / 20220124 / 1000411733214.shtml). Therefore, the yield and safe production of corn directly affect my country's food security and the healthy development of its extended industries.

[0003] Studies have shown that the occurrence and prevalence of corn diseases and insect pests, which cause yield losses and quality decline, are one of the main factors leading to corn production reduction. In recent years, due to the promotion of the straw return system, continuous corn cultivation, and the lack of corn varieties with high resistance to stalk rot in production, the number of pathogenic bacteria in the field has increased, and the damage caused by corn stalk rot has become more serious year by year. The incidence rate is 10-20% in general years and 50-60% in severe years. The pathogens of corn stalk rot are complex, among which Fusarium graminearum and Fusarium pseudoverticillium are the main pathogens of corn Fusarium stalk rot. In addition to harming corn stalks and causing stalk rot, they can also harm corn ears, causing ear mildew and rot, resulting in serious yield losses. In addition, the large amount of fungal toxins they produce also affects the health of humans and animals. At present, fungicides are still the main means of preventing and controlling corn stalk rot, but long-term use of chemical control can easily make crops resistant to drugs, resulting in reduced disease control effects.

[0004] Actinomycetes are a type of microbial resource whose metabolites contain a large number of bioactive substances. The bioactive substances produced by them have the advantages of good activity and low resistance, and have been widely used in agricultural production or as precursor compounds of new fungicides. In addition, the secondary metabolites of actinomycetes are also widely used in steroid transformation, petroleum dewaxing, sewage treatment and biological nitrogen fixation. Jinggangmycin produced by Streptomyces hygroscopicus Jinggangmycin is the most successful biological pesticide developed in my country. It has good control effect on rice sheath blight and long lasting effect. It can effectively control the occurrence of the disease and is now widely used in the prevention and control of rice sheath blight. Zambrano et al. isolated the actinomycete Streptomyces spp.M2A2 from the soil around rice. It can inhibit the growth of rice sheath blight hyphae and delay the onset of symptoms. It can also affect the expansion of pathogens in the susceptible variety Fedearroz 68, and the control effect is equivalent to that of difenoconazole.

[0005] Biological control has many advantages, such as no pollution to the environment, no toxic side effects on crops, and pathogens are not easy to develop resistance. The use of biological control measures to control corn stalk rot is in line with the strategy of sustainable agricultural development. Summary of the invention

[0006] The primary purpose of the present invention is to provide the use of Streptomyces paludis strain 13-3 in preventing and controlling corn stalk rot, in response to the serious occurrence of corn stalk rot, which hinders the healthy development of the corn industry, and the problems of environmental pollution and pathogen resistance caused by the large-scale use of fungicides.

[0007] Another object of the present invention is to provide application of the fermentation liquid of Streptomyces paludis strain 13-3 in preventing and controlling corn stalk rot.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The application of Streptomyces paludis strain 13-3 in the prevention and control of corn stalk rot. The Streptomyces paludis strain 13-3 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit address at No. 1 Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 22830 and the deposit date July 6, 2021.

[0010] The invention discloses an application of a fermentation liquid of a Streptomyces paludis strain 13-3 in preventing and controlling corn stalk rot. The fermentation liquid is prepared by inoculating the Streptomyces paludis strain 13-3 described in claim 1 into an ISP2 solid culture medium, fermenting at a temperature of 26-28° C. for 7-8 days, extracting with ethyl acetate, concentrating the extract under reduced pressure and evaporating to dryness, and then dissolving the extract with DMSO.

[0011] The invention discloses an application of the fermentation liquid of Streptomyces paludis strain 13-3 in inhibiting the hyphae growth of Fusarium graminearum and Fusarium verticillioides.

[0012] The invention discloses an application of the fermentation liquid of Streptomyces paludis strain 13-3 in inhibiting the spore germination of Fusarium graminearum and Fusarium verticillioides.

[0013] Compared with the prior art, the invention has the advantages that the Streptomyces paludis strain 13-3 has an antagonistic effect on two pathogens (Fusarium graminearum and Fusarium verticillioides) that cause corn stalk rot, and its fermentation liquid can significantly inhibit the hyphae growth and spore germination of the two pathogens (Fusarium graminearum and Fusarium verticillioides) that cause corn stalk rot, and the fermentation liquid has an excellent prevention and control effect on corn stalk rot.

[0014] In addition, the Streptomyces paludis strain 13-3 was obtained from soil, is harmoniously compatible with the natural environment, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are the culture characteristics of Streptomyces paludis strain 13-3 on Gao's medium No. 1.

[0016] Figure 2The inhibitory effect of Streptomyces paludis strain 13-3 on Fusarium graminearum and Fusarium verticillioides under confrontation culture conditions. Note: A is Fusarium graminearum, B is Fusarium verticillioides, and CK is the control group.

[0017] Figure 3 The results show that the fermentation liquid of Streptomyces paludis strain 13-3 inhibits the mycelial growth of Fusarium graminearum and Fusarium verticillioides. Note: A is Fusarium graminearum, B is Fusarium verticillioides, and CK is the control group.

[0018] Figure 4 The inhibitory effect of the fermentation liquid of Streptomyces paludis strain 13-3 on the spore germination of Fusarium graminearum and Fusarium verticillioides. Note: A is Fusarium graminearum, B is Fusarium verticillioides, and CK is the control group. DETAILED DESCRIPTION

[0019] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0020] Example 1: Isolation and identification of Streptomyces paludis strain 13-3.

[0021] 1. Collection of soil samples

[0022] Three soil samples were collected from Jinggangshan, Jiangxi Province. The surface soil was removed and soil samples at a depth of 5-20 cm were collected. After marking, the samples were brought back to the laboratory for natural air drying.

[0023] 2. Isolation of actinomycetes

[0024] The air-dried soil sample was ground with a mortar, 1 g of the sample was weighed and suspended in 9 mL of sterile water, shaken at 40°C and 180 rpm for 30 min, and then allowed to stand for 5 min. The sample was diluted 10 times in sequence to prepare 10 -2 , 10 -3 , 10 -40.1 mL of the suspension of different concentrations was taken and added to the modified HVA medium (potassium dichromate with a final concentration of 100-200 ppm) plate. After evenly spreading, the plate was inverted and cultured at 28°C for observation. After 5-7 days, different single colonies were picked out for streak purification. The purified strains were stored in a -80°C refrigerator using the glycerol method.

[0025] 3. Identification of Streptomyces paludis strain 13-3

[0026] (1) Observation of morphological characteristics

[0027] Streptomyces paludis strain 13-3 grew well on most culture media and produced no soluble pigment (Table 1). Under an optical microscope, the spores of strain 13-3 were straight, flexible, hooked, loose and tightly spiral, and the spores were oval and cylindrical.

[0028] Table 1 Cultivation characteristics of Streptomyces paludis 13-3

[0029]

[0030] (2) Physiological and biochemical characteristics

[0031] The starch hydrolysis, nitrate reduction and carbon and nitrogen source utilization characteristics of wetland Streptomyces (Streptomyces paludis) 13-3 were determined according to the method described in the "Streptomyces Identification Manual". The results are shown in Table 2.

[0032] Table 2 Physiological and biochemical characteristics of Streptomyces paludis 13-3

[0033]

[0034]

[0035] (3) Sequence analysis

[0036] After extracting the genomic DNA of Streptomyces paludis 13-3 using a bacterial genome extraction kit, the 16S rRNA and rpoB genes were amplified, and the full-length sequences obtained were 1410bp and 767bp, respectively. The obtained sequences were submitted to the GenBank database for BLAST comparison. The morphological characteristics, physiological and biochemical characteristics, and 16S rRNA and rpoB gene analysis showed that strain 13-3 was Streptomyces paludis.

[0037] Example 2: Determination of the antagonistic effect of Streptomyces paludis strain 13-3 on two pathogens causing corn stalk rot

[0038] The plate confrontation culture method was used to determine the antagonistic effect of Streptomyces paludis strain 13-3 on Fusarium graminearum and Fusarium pseudo-verticillium. First, the strain 13-3 was inoculated on both sides of the PDA medium near the edge. After 3 days, a 5mm diameter bacterial cake of the test pathogen was inoculated in the center of the plate. The plate was cultured at 28°C for 5 days to measure the width of the inhibition zone of Streptomyces paludis strain 13-3 against the test pathogens (Table 3). The test pathogens without antagonistic bacteria were used as controls. The results showed that Streptomyces paludis strain 13-3 had a very strong antagonistic effect on Fusarium graminearum and Fusarium pseudo-verticillium ( Figure 2 ).

[0039] Table 3 Antagonistic effect of strain 13-3 on two types of corn Fusarium stalk rot pathogens

[0040]

[0041] Example 3: Inhibitory effect of the fermentation liquid of Streptomyces paludis strain 13-3 on two pathogens causing corn stalk rot

[0042] The Streptomyces paludis strain 13-3 was activated in Gao's medium No. 1 and cultured at 28°C for 5 days. The spores of the Streptomyces paludis strain 13-3 were inoculated into ISP2 solid medium and fermented at 26-28°C for 7-8 days. The extract was extracted with ethyl acetate for 3 times. The extract was concentrated under reduced pressure and evaporated to dryness, then dissolved with DMSO to prepare a 20 mg / mL fermentation solution for later use.

[0043] Take 100 μL of the fermentation broth of Streptomyces paludis strain 13-3 and add it to 100 mL of PDA medium cooled to about 50°C (not hot), mix well and pour into a culture dish, inoculate a 5 mm diameter cake of the test corn stalk rot pathogen in the center of the PDA medium plate, and culture it at 28°C for 5 days before measuring the colony diameter. Using the addition of the same amount of DMSO as a control, calculate the inhibition rate of the fermentation broth on the mycelial growth of the two corn stalk rot pathogens (Table 4). The results show that the fermentation broth of Streptomyces paludis strain 13-3 has a strong inhibitory effect on the mycelial growth of Fusarium graminearum and Fusarium pseudoverticillium ( Figure 3 ).

[0044] Table 4 Inhibitory effect of the fermentation liquid of strain 13-3 on two types of corn Fusarium stalk rot pathogens

[0045]

[0046] Example 4: Inhibitory effect of the fermentation liquid of Streptomyces paludis strain 13-3 on spore germination of two pathogens causing corn stalk rot

[0047] The 5 mm diameter Fusarium spp. cake was inoculated into PDB liquid culture medium, and the Fusarium graminearum cake was inoculated into mung bean liquid culture medium. After 7 days of shaking culture at 28 °C and 180 rpm, the spore suspension was collected by filtration and its concentration was adjusted to 1 × 10 5 Pieces / mL.

[0048] 1 μL of the fermentation broth of Streptomyces paludis strain 13-3 in Example 3 was added to 1 mL of spore suspension, and the same amount of DMSO was added as a control. The spore suspensions of Fusarium graminearum and Fusarium pseudo-verticillium were cultured at 28°C for 10 h and 4 h, respectively, and the inhibition rate of the fermentation broth of Streptomyces paludis strain 13-3 on the spore germination of two types of corn stalk rot pathogens was calculated (Table 5). The results showed that the fermentation broth of Streptomyces paludis strain 13-3 significantly inhibited the spore germination of Fusarium graminearum and Fusarium pseudo-verticillium ( Figure 4 ).

[0049] Table 5 Inhibitory effect of the fermentation liquid of strain 13-3 on the conidia germination of two types of corn Fusarium stalk rot pathogens

[0050]

[0051] Example 5: Application of the fermentation liquid of Streptomyces paludis strain 13-3 in preventing and controlling corn seedling stalk rot.

[0052] The tested Fusarium graminearum and Fusarium spp. were cultured at 28°C for 5 days, and a 5-mm-diameter puncher was used to punch out bacterial cakes at the edge of the colony. The bacterial cakes were transferred into sterilized wheat grain culture media and cultured in the dark at 28°C for 10 days. Fusarium graminearum and Fusarium spp. were inoculated into sterile soil, that is, the cultured wheat grain culture media were mixed evenly with sterile soil in a mass ratio of 1:20, and placed in nutrient pots for later use.

[0053] The test site was selected in Pudang Village, Jin'an District, Fuzhou City, Fujian Province. Six treatments were set up in the test. The treatments inoculated with Fusarium graminearum were: A. 3% difenoconazole WG 4 ml / kg seeds, B. 10 ml / kg seeds of fermentation liquid of Streptomyces paludis strain 13-3, and C. water control; the treatments inoculated with Fusarium verticillioides were: D. 3% difenoconazole WG 4 ml / kg seeds, E. 10 ml / kg seeds of fermentation liquid of Streptomyces paludis strain 13-3, and F. water control. The treatment agents were measured according to the weight of corn seeds, and the seeds were mixed with each other, spread out and dried, and then sown in nutrient pots, with 10 corn seeds per pot, 5 nutrient pots per treatment, and 4 replicates per treatment.

[0054] When the corn grows to 4-5 leaves, investigate the condition.

[0055] Survey method: Dig up the seedlings and investigate the disease conditions of corn plants and roots according to the following grading standards:

[0056] Level 0: The whole plant grows normally and is disease-free;

[0057] Level 1: The plant grows basically normally, but a small amount of lesions can be seen on the roots, and the area of ​​lesions accounts for less than 1 / 4 of the root surface area. The root color is white with brown.

[0058] Grade 2: The plant grows weakly, the leaf color becomes lighter, the plant height becomes shorter, only 3 / 4 of the control, the lateral roots are few and short, there are no hairy roots, the lesions are continuous, and the area of ​​lesions accounts for 1 / 4 to 1 / 2 of the total root surface area. The color of the root group is white and brown.

[0059] Grade 3: The plant grows very abnormally, with the aboveground part showing signs of withering or yellowing, the plant height is only 1 / 2 of the control, the lateral roots are very few, the area of ​​lesions accounts for 1 / 2 to 3 / 4 of the total root surface area, and the root color is brown with white.

[0060] Level 4: The seeds germinate but do not emerge, are almost suffocated to death, are covered with lesions, and the roots are brown;

[0061] Disease index = ∑ (number of diseased plants at each level × relative disease level value) ÷ (total number of plants investigated × 4) × 100;

[0062] Control effect (%) = [(CK disease index - treatment disease index) ÷ CK disease index] × 100;

[0063] Table 6 Control effect of the fermentation liquid of strain 13-3 on corn Fusarium stalk rot

[0064]

[0065] The experimental results show that the control effects of A.3% difenoconazole WG 4 ml / kg seeds and B. fermentation liquid of Streptomyces paludis strain 13-3 10 ml / kg seeds on corn stem rot caused by Fusarium graminearum are 66.46% and 63.90% respectively, which are safe for corn and no phytotoxicity is observed. The control effect of fermentation liquid of Streptomyces paludis strain 13-3 10 ml / kg seeds on corn stem rot caused by Fusarium graminearum is not significantly different from that of 3% difenoconazole WG 4 ml / kg seeds at the 5% significant level and the 1% extremely significant level.

[0066] D. 3% difenoconazole WG 4 ml / kg seed and fermentation liquid of Streptomyces paludis strain 13-3 10 ml / kg seed had a protective effect of 63.57% and 61.23% respectively on corn stem rot caused by Fusarium verticillioides, both of which were safe for corn and no phytotoxicity was observed. The protective effect of fermentation liquid of Streptomyces paludis strain 13-3 10 ml / kg seed on corn stem rot caused by Fusarium verticillioides was not significantly different from that of 3% difenoconazole WG 4 ml / kg seed at the 5% significant level and the 1% extremely significant level.

[0067] It can be seen that the fermentation liquid of Streptomyces paludis strain 13-3 at 10 ml / kg seed has a comparable control effect against corn Fusarium stalk rot as 3% difenoconazole WG at 4 ml / kg seed, and has good application prospects.

Claims

1. Streptomyces limnifolia ( Streptomyces marsh ) The use of strain 13-3 in preventing and controlling corn stalk rot is characterized by: The wetland Streptomyces ( Streptomyces marsh ) strains 13-3 were respectively composed of Fusarium graminearum ( Fusarium grasses ) and Pseudomonas verticillasters ( Fusarium verticillium ) has a preventive and therapeutic effect on corn stalk rot caused by The wetland Streptomyces ( Streptomyces marsh ) strain 13-3 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 22830 and the deposit date: July 6, 2021.

2. Streptomyces limnifolia ( Streptomyces marsh ) Application of the fermentation liquid of strain 13-3 in preventing and treating corn stalk rot, characterized in that: The wetland Streptomyces ( Streptomyces marsh ) strain 13-3 fermentation broth was respectively composed of Fusarium graminearum ( Fusarium gramineae ) and Pseudomonas verticillasters ( Fusarium verticillium ) has a preventive and therapeutic effect on corn stalk rot caused by The fermentation broth is prepared by mixing the Streptomyces palustris described in claim 1 ( Streptomyces marsh ) strain 13-3 was inoculated into ISP2 solid culture medium, the fermentation temperature was 26-28°C, the fermentation culture time was 7-8 d, the extraction was performed with ethyl acetate, the extract was concentrated under reduced pressure and evaporated to dryness, and then dissolved with DMSO to obtain the product.

3. The use according to claim 2, characterized in that: The wetland Streptomyces ( Streptomyces marsh ) strain 13-3 fermentation broth in inhibiting Fusarium graminearum ( Fusarium gramineae ) and Pseudomonas verticillasters ( Fusarium whorl-shaped ) in the application of mycelial growth.

4. The use according to claim 2, characterized in that: The wetland Streptomyces ( Streptomyces marsh ) strain 13-3 fermentation broth in inhibiting Fusarium graminearum ( Fusarium gramineae ) and Pseudomonas verticillasters ( Fusarium whorl-shaped ) in the spore germination.

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