New application of a strain of conidiobolus obscurus in preventing and treating sclerotinia sclerotiorum

Abstract

A new application of a pyrenochaeta nobilis strain in preventing and treating sclerotinia, belongs to the field of agricultural microbial technology. In order to screen the antagonistic strain of sclerotinia, and then provide the core microbial resources for the environment-friendly and sustainable biological control technology of sclerotinia, through the plate confrontation experiment, the indoor sclerotinia sclerotium germination experiment, the sunflower field sclerotinia sclerotium germination experiment and the industrial hemp sclerotinia pot experiment, it is found that the pyrenochaeta nobilis SFJ12-R-5 has the inhibitory effect on the mycelium growth of sclerotinia, and the fermentation liquor of the pyrenochaeta nobilis SFJ12-R-5 can inhibit the sclerotinia sclerotium germination and has the prevention and treatment effect on the sclerotinia, and can be used for preventing and treating the sunflower sclerotinia and the industrial hemp sclerotinia.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a new application of a strain of Echinococcus in the control of Sclerotinia sclerotiorum. Background Technology

[0002] Sclerotinia sclerotiorum, a fungus causing sclerotinia rot, is a significant, globally distributed disease. This fungus can infect over 450 plant species, including important economic crops such as rapeseed, soybean, lettuce, and sunflower. Sclerotinia sclerotiorum can infect various parts of crops and has a wide host range. The fungus forms sclerotia, allowing it to survive persistently in the soil through winter and summer. Under suitable temperature and humidity conditions, the sclerotia germinate, producing apothecia, and the released ascospores cause primary infection.

[0003] Currently, the control of Sclerotinia sclerotinia is still mainly based on chemical pesticides. However, the long-term and repeated use of chemical pesticides also threatens crop safety and the ecological environment. At this point, biological control shows its advantages. Effective biological control can reduce the amount of chemical pesticides used, reduce environmental pollution, and improve crop resistance while controlling the growth and reproduction of pathogens. Currently, only two fungicides have been approved and registered in my country for the control of Sclerotinia sclerotinia, with active ingredients *Coniothyrium minitans* CGMCC8325 and *Coniothyrium sp.* ZS1SB, respectively. Therefore, identifying antagonistic strains of *Sclerotinia sclerotinia* to provide core microbial resources for environmentally friendly and sustainable biological control technologies for Sclerotinia sclerotinia is an urgent problem to be solved. Summary of the Invention

[0004] In order to screen out antagonistic strains of Sclerotinia sclerotiorum and thus provide core microbial resources for environmentally friendly and sustainable biological control of Sclerotinia sclerotiorum, this invention provides a new application of Pyrenochaeta nobilis in the control of Sclerotinia sclerotiorum. This strain can inhibit the mycelial growth of Sclerotinia sclerotiorum, and its fermentation broth can inhibit the germination of Sclerotinia sclerotia. It can also be applied to the control of Sclerotinia sclerotiorum in sunflowers and industrial hemp.

[0005] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution:

[0006] The first objective of this invention is to provide the application of a strain of Echinococcus SFJ12-R-5 in inhibiting the mycelial growth of Sclerotinia sclerotiorum. This Echinococcus SFJ12-R-5 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC NO.17766, deposit date May 31, 2019, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. This strain has been disclosed in patent application number 201910834356.8, entitled "An Echinococcus SFJ12-R-5 from Astragalus membranaceus roots that effectively inhibits tomato gray mold and its application."

[0007] The second objective of this invention is to provide the application of the fermentation broth of the above-mentioned Echinococcus SFJ12-R-5 in inhibiting the germination of Sclerotium sclerotia.

[0008] In one embodiment of the present invention, the fermentation broth of *Echinococcus spp.* SFJ12-R-5 is obtained by inoculating *Echinococcus spp.* SFJ12-R-5 onto a sporulation medium, culturing at 24–26°C for 5 days, then placing 7 mm diameter microspheres at the edge of colonies covering petri dishes, inoculating three microspheres into a fermentation broth, and culturing at 24–26°C and 180 r / min on a shaker for 10 days. The fermentation broth consists of 40 g / L glucose, 10 g / L peptone, 4 g / L KH₂PO₄, 0.3 g / L MgSO₄·7H₂O, 0.1 g / L FeSO₄·7H₂O, with the remainder being water.

[0009] The third objective of this invention is to provide the application of the fermentation broth of the above-mentioned Echinococcus SFJ12-R-5 in the prevention and control of sclerotinia rot in plants.

[0010] In one embodiment of the invention, the plant is a sunflower or industrial hemp.

[0011] In one embodiment of the present invention, the fermentation broth of *Echinococcus spp.* SFJ12-R-5 is obtained by inoculating *Echinococcus spp.* SFJ12-R-5 onto a sporulation medium, culturing at 24–26°C for 5 days, then placing 7 mm diameter microspheres at the edge of colonies covering petri dishes, inoculating three microspheres into a fermentation broth, and culturing at 24–26°C and 180 r / min on a shaker for 10 days. The fermentation broth consists of 40 g / L glucose, 10 g / L peptone, 4 g / L KH₂PO₄, 0.3 g / L MgSO₄·7H₂O, 0.1 g / L FeSO₄·7H₂O, with the remainder being water.

[0012] In one embodiment of the present invention, the above-mentioned sporulation culture medium is SADY medium, which consists of 10 g / L peptone, 40 g / L glucose, 10 g / L yeast extract, 20 g / L agar, and the remainder is water.

[0013] The fourth objective of this invention is to provide an application of a microbial fertilizer containing the aforementioned Echinococcus SFJ12-R-5 in the prevention and control of sclerotinia rot in plants.

[0014] The fifth objective of this invention is to provide an inoculum containing the fermentation broth of *Echinochloa crus-galli* SFJ12-R-5 for the prevention and control of sclerotinia rot in plants.

[0015] In one embodiment of the invention, the plant is a sunflower or industrial hemp.

[0016] The beneficial effects of this invention are:

[0017] This invention, through plate confrontation experiments, indoor sclerotium germination experiments, sunflower field sclerotium germination experiments, and pot experiments on sclerotium rot in industrial hemp, found that Pyrenochaeta nobilis SFJ12-R-5 has an inhibitory effect on the mycelial growth of Pyrenochaeta nobilis, and that the fermentation broth of Pyrenochaeta nobilis SFJ12-R-5 can inhibit the germination of Pyrenochaeta nobilis sclerotia, thus having a preventive effect on sclerotium rot and can be used to control sclerotium rot in sunflowers and industrial hemp. Attached Figure Description

[0018] Figure 1 The image shows the inhibitory effect of Echinococcus SFJ12-R-5 on Sclerotinia sclerotiorum hyphae; among them, Figure 1 In the diagram, A represents the experimental group where Echinococcus SFJ12-R-5 confronted Sclerotinia. Figure 1 B in the table represents the control group that was inoculated with only Sclerotinia sclerotiorum;

[0019] Figure 2 The image shows the inhibitory effect of Echinococcus SFJ12-R-5 on the sclerotium germination of Sclerotium sclerotia; among them, Figure 2 In the diagram, A represents the water control group. Figure 2 In the diagram, B represents the treatment group with a 50-fold dilution of Echinococcus SFJ12-R-5 fermentation broth. Figure 2 C in the text refers to the treatment group of 500-fold dilution of Echinococcus SFJ12-R-5 fermentation broth;

[0020] Figure 3 The image shows the inhibitory effect of Echinococcus SFJ12-R-5 fermentation broth on the sclerotium germination of Sclerotium sclerotiorum in sunflower fields; among them, Figure 3 In the diagram, A represents the water control group. Figure 3 In the diagram, B represents the treatment group with a 50-fold dilution of Echinococcus SFJ12-R-5 fermentation broth. Figure 3 C in the text refers to the treatment group of 500-fold dilution of Echinococcus SFJ12-R-5 fermentation broth;

[0021] Figure 4The image shows the control effect of Echinococcus SFJ12-R-5 fermentation broth on sclerotinia rot in industrial hemp in a pot experiment; among them, Figure 4 In the diagram, A represents the water control group. Figure 4 In the diagram, group B represents the treatment group that received only a suspension of sclerotinia stem rot pathogens. Figure 4 In the figure, C represents the treatment group that was treated with a suspension of Sclerotinia sclerotiorum and then sprayed with a 50-fold dilution of Echinococcus SFJ12-R-5 fermentation broth. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0023] The *Pyrenochaeta nobilis* SFJ12-R-5 used in this invention has been deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC NO.17766, deposit date May 31, 2019, address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. This strain has been disclosed in patent application number 201910834356.8, entitled "A Highly Effective *Pyrenochaeta nobilis* from Astragalus Roots for Inhibiting Tomato Gray Mold and Its Application".

[0024] The Sclerotinia sclerotiorum used in this invention is preserved at the Institute of Plant Protection, Heilongjiang Academy of Agricultural Sciences.

[0025] The liquid culture medium used in this invention has the following composition: glucose 40 g / L, peptone 10 g / L, KH2PO4 4 g / L, MgSO4·7H2O 0.3 g / L, FeSO4·7H2O 0.1 g / L, and the balance is water.

[0026] The composition of the SADY sporulation medium used in this invention is as follows: 10g peptone, 40g glucose, 10g yeast extract, 20g agar to 1L water.

[0027] The preparation method of the fermentation broth of Echinococcus SFJ12-R-5 used in this invention is as follows: Activate the slant of Echinococcus SFJ12-R-5 stored at 4℃, transfer it to the sporulation medium SADY, and incubate in the dark in an artificial intelligence incubator at 24-26℃ for 5 days, ready for use; make several 7mm bacterial discs at the edge of the colony that has covered the petri dish, inoculate the bacterial discs into Erlenmeyer flasks containing 300mL of sterile liquid culture medium, inoculate 3 bacterial discs into each flask, and incubate in a shaker at 24-26℃ and 180r / min for 10 days, and retain the fermentation broth for experiments.

[0028] Example 1: Inhibitory effect of Echinococcus SFJ12-R-5 on Sclerotinia sclerotiorum.

[0029] Echinococcus SFJ12-R-5 and the pathogen (Sclerotinia sclerotiorum) preserved on activated slant culture were transferred to SADY plates for plate confrontation experiments. The specific experimental method is as follows: Using a 5mm diameter punch, bacterial discs were made at the edges of both Sclerotinia sclerotiorum and Echinococcus SFJ12-R-5. One Sclerotinia sclerotiorum and one Echinococcus SFJ12-R-5 disc were inoculated along the diameter of a 90mm diameter SADY plate, 1cm from the edge. Three plates were repeated for each treatment. Plates inoculated only with Sclerotinia sclerotiorum served as controls. The plates were incubated in the dark at 25℃. Once the control colonies had fully colonized the plates, the colony radius of the treated Sclerotinia sclerotiorum was measured with a ruler, and the mycelial growth inhibition rate (IMG) was calculated. IMG = (Control colony radius - Treated pathogen colony radius) / Control colony radius × 100%.

[0030] The results of the standoff experiment are as follows Figure 1 As shown, confrontation culture revealed that Echinococcus SFJ12-R-5 has an inhibitory effect on Sclerotinia sclerotiorum, with a growth inhibition rate of 28.57%, indicating that Echinococcus SFJ12-R-5 can inhibit the growth of Sclerotinia sclerotiorum hyphae.

[0031] Example 2: Inhibitory effect of Echinococcus SFJ12-R-5 fermentation broth on germination of Sclerotium sclerotia in Sclerotium sclerotia

[0032] Sterilized soil to a depth of approximately 5 cm was placed in germination boxes, and sterile water was used to maintain soil moisture at approximately 60%. Twenty *Sclerotium sclerotia* sclerotia were placed in each germination box. The sclerotia were treated with 50-fold and 500-fold dilutions of the *Echinococcus spp.* SFJ12-R-5 fermentation broth prepared according to this invention, with water serving as a blank control. Each box was sprayed with 10 mL of the treatment solution, and each treatment was repeated three times. The germination boxes were placed in an artificial intelligence climate incubator with an L:D ratio of 12:12 and a temperature of 20℃. After 45 days, the germination of the sclerotia in each treatment was observed, and the germination rate of the water control group and the germination inhibition rate of the treatment groups were calculated.

[0033] Sclerotium germination rate (%) = (Number of germinating sclerotia / Total number of sclerotia) × 100%

[0034] Sclerotium germination inhibition rate (%) = (1 - number of germinating sclerotia / total number of sclerotia) × 100%

[0035] The results of the sclerotium germination experiment of Sclerotium sclerotium are shown in Figure 2 In the water-treated blank control, most sclerotia had germinated, and each sclerotia produced multiple ascospore stalks (see...). Figure 2 (A) In the treatment with a 50-fold dilution of Echinococcus SFJ12-R-5 fermentation broth, almost all sclerotia failed to germinate (see A). Figure 2 (B in the text); After treatment with a 500-fold dilution of Echinococcus SFJ12-R-5 fermentation broth, a few sclerotia were observed to germinate (see B in the text). Figure 2 (C) By calculating the sclerotium germination rate, it can be seen that the sclerotium germination rate in the water control group reached 81.67%, the sclerotium germination inhibition rate in the 50-fold dilution of Echinococcus SFJ12-R-5 fermentation broth treatment group reached 100%, and the sclerotium germination inhibition rate in the 500-fold dilution of Echinococcus SFJ12-R-5 fermentation broth treatment group was 63.36%. It can be seen that the 50-fold dilution of Echinococcus SFJ12-R-5 fermentation broth can significantly inhibit the germination of Sclerotium sclerotia.

[0036] Example 3: Inhibitory effect of Echinococcus SFJ12-R-5 fermentation broth on sclerotium germination of Sclerotium sclerotiorum in sunflower fields

[0037] The experimental area was located in the Democratic Experimental Park of the Heilongjiang Academy of Agricultural Sciences. Sunflowers were sown in hills on ridges with a plant spacing of 60 cm and a ridge spacing of 65 cm, and normal field management was implemented. Ten days after sowing, seedlings were placed at fixed points between the ridges, each with an area of ​​0.08 m². 2 An enclosure was constructed (to prevent rainwater from washing away the sclerotia), and 50 *Sclerotium sclerotia* sclerotia were evenly placed inside the enclosure. The experiment was conducted with two treatment times: one on the day the sclerotia were placed in early June, and another in late July when sclerotia germination first appeared in the field. The experimental treatments were consistent with the indoor germination box experiment, with each treatment replicated three times. All treatments were sprayed onto the surface soil inside the enclosure. After the initial sclerotia germination, the germination status was observed every 2-3 days. The inhibitory effect of *Echinococcus spp.* SFJ12-R-5 fermentation broth on the germination of *Sclerotium spp.* sclerotia in the sunflower field was shown in the figure on August 4th. Figure 3 The statistical results of sclerotium germination rate at different time points are shown in Table 1. Figure 3 As shown in Figure A, in the water control group, most sclerotia germinated and produced yellow apothecia; Figure 3 As shown in B, the sclerotia treated with a 50-fold dilution of Echinococcus SFJ12-R-5 fermentation broth hardly germinated, indicating that the fermentation broth significantly inhibited the germination of Sclerotinia sclerotia. Figure 3 As shown in C, a small number of sclerotia treated with a 500-fold dilution of Echinococcus SFJ12-R-5 fermentation broth produced yellow apothecia.

[0038] Table 1. Cumulative field germination rate (%) of sclerotia at different time points after initial germination of each treatment.

[0039]

[0040] Example 4: The effect of Echinococcus SFJ12-R-5 fermentation broth on the control of Sclerotinia sclerotinia in industrial hemp in a pot experiment.

[0041] Healthy industrial hemp plants were selected, with three plants per treatment group. A water control was included, and the treatment group was sprayed with 5 mL of a solution at a concentration of 2.0 × 10⁻⁶. 7 Three treatments were administered: a spore / L suspension of Sclerotinia sclerotiorum pathogen, and a treatment involving spraying 5 mL of Sclerotinia sclerotiorum pathogen for 1 hour followed by a 50-fold dilution of 5 mL of Echinococcus spp. SFJ12-R-5 fermentation broth. Each treatment was sprayed onto the surface of the industrial hemp plants. After treatment, the potted plants were covered with plastic bags to maintain humidity and placed in an AI climate incubator with an L:D ratio of 12:12 and a temperature of 20°C. Disease incidence was assessed after 6 days (see [link to relevant documentation]). Figure 4 ).like Figure 4 As shown in Figure A, the industrial hemp plants in the water control group grew healthily; as... Figure 4 As shown in B, in the group treated with the sclerotinia rot pathogen, the leaves initially showed water-soaked appearance, and wet rot was clearly visible on the lower part of the plant, with a disease index of 61.11; Figure 4 As shown in Figure C, in the group treated with a 50-fold dilution of Echinococcus SFJ12-R-5 fermentation broth, a few leaves showed typical "V"-shaped lesions at the leaf tips, with a disease index of 16.67. The 50-fold dilution of Echinococcus SFJ12-R-5 fermentation broth achieved a control efficacy of 72.72% against Sclerotinia sclerotinia in potted industrial hemp.

[0042] The grading standard for sclerotinia stem rot in industrial hemp is based on the grading standard for sclerotinia stem rot in sunflowers, and is divided into 5 levels: Level 0 indicates no disease; Level 1 indicates that the lesion area accounts for less than 10% of the whole leaf; Level 2 indicates that the lesion area accounts for 10% to 30% of the whole leaf; Level 3 indicates that the lesion area accounts for 31% to 50% of the whole leaf; and Level 4 indicates that the lesion area accounts for more than 51% of the whole leaf.

[0043] Disease index = [∑(number of diseased plants × representative level) / (total number of plants × highest representative value)] × 100

[0044] Prevention and control efficacy (%) = (Disease index of pathogen control group - Disease index of treatment group) / Disease index of pathogen control group × 100

[0045] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A strain of Echinococcus ( Pyrenochaeta nobilis SFJ12-R-5 inhibits Sclerotinia sclerotiorum (Sclerotinia sclerotiorum). Sclerotinia sclerotiorum Its application in mycelial growth, characterized in that, The preservation number of Echinococcus SFJ12-R-5 is CGMCCNO. 17766.

2. The application of the fermentation broth of Echinococcus SFJ12-R-5 as described in claim 1 in inhibiting the germination of Sclerotium sclerotia.

3. The application according to claim 2, characterized in that, The fermentation broth of *Echinococcus spp.* SFJ12-R-5 was obtained by inoculating *Echinococcus spp.* SFJ12-R-5 onto a sporulation medium and culturing it at 24-26℃ for 5 days. Then, colonies that had grown on petri dishes were inoculated onto fermentation medium and cultured on a shaker at 24-26℃ and 180 r / min for 10 days. The fermentation medium consisted of 40 g / L glucose, 10 g / L peptone, 4 g / L KH2PO4, 0.3 g / L MgSO4·7H2O, 0.1 g / L FeSO4·7H2O, with the remainder being water.

4. The use of the fermentation broth of Echinococcus SFJ12-R-5 as described in claim 1 in the prevention and control of sclerotinia disease in sunflowers or industrial hemp caused by Sclerotinia sclerotiorum.

5. The application according to claim 4, characterized in that, The fermentation broth of *Echinococcus spp.* SFJ12-R-5 was obtained by inoculating *Echinococcus spp.* SFJ12-R-5 onto a sporulation medium and culturing it at 24-26℃ for 5 days. Then, colonies that had grown on petri dishes were inoculated onto fermentation medium and cultured on a shaker at 24-26℃ and 180 r / min for 10 days. The fermentation medium consisted of 40 g / L glucose, 10 g / L peptone, 4 g / L KH2PO4, 0.3 g / L MgSO4·7H2O, 0.1 g / L FeSO4·7H2O, with the remainder being water.

6. The application according to claim 3 or claim 5, characterized in that, The sporulation medium is SADY medium, which consists of 10 g / L peptone, 40 g / L glucose, 10 g / L yeast extract, 20 g / L agar, and the remainder is water.

7. The use of a microbial fertilizer containing Echinococcus SFJ12-R-5 as described in claim 1 in the prevention and control of sclerotinia disease in sunflowers or industrial hemp caused by Sclerotinia sclerotiorum.

8. The use of a microbial agent containing the fermentation broth of Echinococcus SFJ12-R-5 as described in claim 1 in the prevention and control of sclerotinia disease in sunflowers or industrial hemp caused by Sclerotinia sclerotiorum.

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