Rice endophytic cladosporium cladosporium GLARF-7 as well as biological preparation and application thereof in prevention and treatment of rice blast

Abstract

The invention discloses a rice endophytic cladosporium cladosporium GLARF-7, a biological preparation thereof and application of the rice endophytic cladosporium cladosporium GLARF-7 in prevention and treatment of rice blast, and belongs to the technical field of biopesticides. The cladosporium cladosporioides GLARF-7 disclosed by the invention is preserved in the Guangdong Microbial Culture Collection Center on December 31, 2024, and the preservation number of the cladosporium cladosporioides GLARF-7 is GDMCC No: 65694; the fermentation liquor or the supernate of the cladosporium cladosporioides GLARF-7 is used for preparing a corresponding biological agent. The cladosporium cladosporioides GLARF-7 and the biological preparation thereof are applied to prevention and treatment of rice blast. The biocontrol agent is used for inhibiting normal growth of magnaporthe oryzae and has an excellent biocontrol effect of preventing and treating the magnaporthe oryzae, fermentation liquor or supernate of the biocontrol agent is sprayed to the surfaces of rice tissues by controlling the biomass of the biocontrol agent to be used for preventing and treating the magnaporthe oryzae, and the influence on the ecological environment is small; the strain is low in culture condition requirement and has good development and application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological pesticides, and particularly relates to an endophytic Cladosporium cladosporioides GLARF-7 of rice, its biological agent and its application in controlling rice blast disease. Background Art

[0002] Rice (Oryza sativa L.) is an important food crop in the world, and the safe production of rice directly affects food security. Rice blast disease (Magnaporthe oryzae) is one of the most serious fungal diseases in rice production. This disease can break out in all rice planting areas and at different growth stages of rice, with the characteristics of wide distribution and great harm. Moreover, it seriously affects the yield and quality of rice, and may even lead to a complete crop failure in severe cases. Traditionally, the control of rice blast disease mainly relies on chemical control. Although chemical control has a quick effect, good results and plays a key role in controlling the current rice blast pathogen, it will cause certain pollution to the surrounding environment and is very likely to lead to the resistance of rice blast pathogens to it. In addition, the rice blast pathogen mutates rapidly, resulting in a decline in the disease resistance of varieties and the control effect of pesticides. Therefore, studying beneficial biocontrol bacteria for rice blast disease, and using microbial pesticides prepared from biocontrol bacteria can effectively reduce the toxicity and residues of conventional pesticides. It is a safe, efficient and environmentally friendly pesticide, which is of great significance for controlling the amount of agricultural residues, reducing the occurrence of rice blast disease and ensuring the safe production of rice.

[0003] Endophytic fungi are a type of fungi that colonize the internal organs, tissues, and intercellular spaces of plants without causing obvious symptoms in the plants and have a stable living space in the host plants. In most cases, endophytic fungi can obtain the nutrients they need for growth from the host plants, and the secondary metabolites synthesized during the growth process can also enhance the stress resistance of the plants. The secondary metabolites they produce are an important resource for new drug research and development. Liu Zhengqiong et al. found that the secondary metabolites produced by endophytic fungi isolated from Houttuynia cordata have antibacterial activities against Fusarium graminearum, Alternaria solani, Phytophthora capsici, and Fusarium oxysporum f. sp. niveum. Lin et al. isolated an endophytic fungus Diaporthe sp. from citrus, which has a significant inhibitory effect on the growth of Xanthomonas citri subsp. citri and can effectively control citrus canker. Zhao et al. found through research that the endophytic fungus Seimatosporium sp. can improve the ability of roses to resist powdery mildew. Nassim et al. found that the endophytic fungus Piriformospora indica can increase plant biomass, delay the infection process of Rhizoctonia solani, reduce the severity of sheath blight, and have a very significant defensive effect on wheat sheath blight. Su Qin et al. isolated and screened a strain of Cladosporium sp., which can effectively inhibit banana wilt while promoting the growth of bananas. Although a few Cladosporium sp. are pathogens of plants, the low-abundance Cladosporium sp. in the body under natural conditions does not cause disease in well-growing rice plants, and the secondary metabolites of this fungus have significant inhibitory effects on a variety of plant pathogenic fungi and bacteria.

[0004] In summary, Cladosporium sp., as an important natural resource, has attracted wide attention and has the potential to develop biological pesticides.

[0005] At present, there are few reports on the research and utilization of Cladosporium sp. as a biocontrol agent, and most of them focus on its inhibitory effects on rust, banana wilt pathogen, brown spot pathogen, sheath rot pathogen, Burkholderia glumae, Botrytis cinerea of broad bean, and Phytophthora capsici. There are few reports on its inhibitory effect on Magnaporthe oryzae. Summary of the Invention

[0006] The object of the present invention is to provide a strain of endophytic Cladosporium cladosporioides GLARF-7 in rice, its biological agent, and its application in controlling rice blast, aiming to solve the problems of pesticide toxicity and residue existing in the existing pesticides for controlling rice blast. The Cladosporium cladosporioides GLARF-7 in the present invention is isolated from the root tissue of the rice variety Guangluai 4, and can colonize well in rice tissues; it has a significant inhibitory effect on the growth of Magnaporthe oryzae, and has excellent potential biocontrol effect on controlling rice blast; this bacterium is an endophytic fungus in rice roots, and its source is environmentally friendly and non-toxic. It can not only inhibit the growth of Magnaporthe oryzae, but also has little impact on the ecological environment, and is safer and more reliable for the consumption of rice and its processed products; the culture conditions are less demanding, and it has good development and application prospects.

[0007] The object of the present invention is achieved through the following technical solutions:

[0008] A strain of endophytic Cladosporium cladosporioides GLARF-7 in rice, the Cladosporium cladosporioides GLARF-7 was deposited at the Guangdong Provincial Culture Collection of Microorganisms on December 31, 2024. The deposit address is the 5th Floor of Building 59, No. 100, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. The deposit number of the Cladosporium cladosporioides GLARF-7 is GDMCC No: 65694.

[0009] Furthermore, the above-mentioned Cladosporium cladosporioides GLARF-7 is applied in controlling rice blast.

[0010] Furthermore, the Cladosporium cladosporioides GLARF-7 is isolated and purified from rice roots. When the endophytic Cladosporium cladosporioides GLARF-7 in rice is cultured on a PDA medium at 28 °C for 7 days, the colony is villous, brownish-green, grows flat in a circular shape, the surface is rough and opaque, and the back is dark green.

[0011] More preferably, the rice variety used for the isolation and purification is Guangluai 4.

[0012] Another object of the present invention is to provide a biological agent of endophytic Cladosporium cladosporioides GLARF-7 in rice, and the biological agent is prepared based on the above-mentioned Cladosporium cladosporioides GLARF-7.

[0013] Furthermore, the biological agent of endophytic Cladosporium cladosporioides GLARF-7 in rice is the fermentation broth of Cladosporium cladosporioides GLARF-7 or the supernatant of Cladosporium cladosporioides GLARF-7.

[0014] Furthermore, the preparation method of the Cladosporium cladosporioides GLARF-7 fermentation broth specifically includes the following steps: inoculate Cladosporium cladosporioides GLARF-7 into a liquid PD medium and shake culture for 7 days to obtain the Cladosporium cladosporioides GLARF-7 fermentation broth.

[0015] Furthermore, the preparation method of the supernatant of Cladosporium cladosporioides GLARF-7 specifically includes the following steps: inoculate Cladosporium cladosporioides GLARF-7 into a liquid PD medium and shake culture for 7 days, then centrifuge at 10,000 rpm for 1 minute to take the supernatant to obtain the supernatant of Cladosporium cladosporioides GLARF-7.

[0016] Furthermore, the pH value of the liquid PD medium is 7.0.

[0017] Furthermore, the specific conditions for the shake culture for 7 days are: the liquid PD medium is cultured at 28 - 30 °C with a shaker oscillation rate of 180 - 200 rpm for 7 days. The present invention also provides the application of the above-mentioned endophytic Cladosporium cladosporioides GLARF-7 in rice in the prevention and control of rice blast.

[0018] Combined with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0019] First, the Cladosporium cladosporioides GLARF-7 obtained in the present invention is derived from the rice roots and can colonize well in the rice tissues; in addition, the antagonistic endophyte screened from the rice is used for the prevention and control of rice blast, which only shows the increase or decrease of the number of certain microorganisms among specific populations and does not change the category of the microbial population on the rice so as to affect the stability of the population. In addition, due to its long-term coexistence and mutualistic symbiosis with the rice, a series of survival mechanisms similar to it have been formed. Spraying it on the surface of the rice tissues for disease prevention and control is safer and more reliable for the consumption of rice and its processed products.

[0020] Second, the Cladosporium cladosporioides GLARF-7 of the present invention has a strong inhibitory effect on the rice blast pathogen, has excellent potential biocontrol effects on preventing and controlling rice blast, and is environmentally friendly in biological sources and has little impact on the ecological environment. The Cladosporium cladosporioides obtained in the present invention has low requirements for culture conditions and has good development and application prospects.

[0021] Third, the expected benefits and commercial values after the transformation of the technical solutions of the present invention are: based on the significant inhibitory effect of the microbial resources to be protected by this patent on the growth of the rice blast pathogen, researching and developing biogenic pesticides based on this bacterium for the prevention and control of rice blast can effectively reduce the use of chemical pesticides, be environmentally friendly, ensure food security, have good ecological benefits, and promoting the development of the bacterial agent based on this bacterium can obtain economic benefits and commercial values.

[0022] Fourth, the present invention further enriches the microbial resources related to biological control research at home and abroad. By using different treatment methods, different microbial agents can be prepared, solving the technical problem of the single use of microbial pesticides. Through different treatments, the present invention can be used to control rice blast, and can also be mixed with other microorganisms to stabilize the microbial community in the rice growth environment, promote the healthy growth of rice and improve the horizontal disease resistance of rice.

[0023] Fifth, as an endophyte of rice, the present invention overcomes the technical prejudice of the single action of general biological pesticides. The present invention can not only be used as an important means to control rice blast, but also stabilize the microbial community of rice itself, contribute to the growth and development of rice, and create a more suitable microbial environment for the survival of rice. Brief Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments of the present invention. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1 It is a physical diagram of Cladosporium cladosporioides GLARF-7 provided by the embodiment of the present invention on PDA medium; wherein, A is the front view and B is the back view;

[0026] Figure 2 It is an effect diagram of the inhibitory effect of Cladosporium cladosporioides GLARF-7 provided by the embodiment of the present invention on Magnaporthe oryzae; wherein, A is the colony morphology of Magnaporthe oryzae growing normally, B is the colony morphology of Cladosporium cladosporioides GLARF-7 growing normally, and C is the inhibition diagram of Cladosporium cladosporioides GLARF-7 on Magnaporthe oryzae;

[0027] Figure 3 It is an effect diagram of the control of Magnaporthe oryzae by the biological agent of Cladosporium cladosporioides GLARF-7 provided by the embodiment of the present invention; wherein, A is the colony morphology of Magnaporthe oryzae growing normally, B is the inhibition diagram of the fermentation broth of Cladosporium cladosporioides GLARF-7 on Magnaporthe oryzae, C is the inhibition diagram of the supernatant of Cladosporium cladosporioides GLARF-7 on Magnaporthe oryzae, and D is the inhibition diagram of the sterile filtrate of Cladosporium cladosporioides GLARF-7 on Magnaporthe oryzae. Detailed Embodiments

[0028] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] In addition, the technical solutions between various embodiments can be combined with each other, but it must be based on the fact that those of ordinary skill in the art can implement them. When the combination of technical solutions results in contradictions or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the protection scope required by the present invention.

[0030] At present, there are problems of pesticide toxicity and residue in existing pesticides for controlling rice blast. To solve the above technical problems, the present invention provides a strain of endophytic Cladosporium cladosporioides GLARF-7 in rice, its biological agent, and its application in controlling rice blast.

[0031] Example 1: Isolation, purification and preservation of endophytic Cladosporium cladosporioides GLARF-7 in rice roots

[0032] (1) Preparation of liquid PD medium: Weigh 200 g of potatoes, wash, peel and chop them, add 1000 mL of water and boil for 0.5 h, filter with gauze, then add 20 g of glucose (if preparing PDA medium, add another 20 g of agar), dissolve thoroughly, sterilize at 121 °C for 20 min, dispense into petri dishes, cool and store for later use.

[0033] (2) Isolation and purification of endophytic Cladosporium cladosporioides in rice roots:

[0034] The rice sample for isolation is the rice variety Guangluai No. 4. Take the root tissues of plants grown in a rice blast nursery that show disease resistance and health. Refer to the conventional tissue isolation method and make a slight modification: Wash the root tissues thoroughly with tap water, dry them, and then perform surface disinfection according to the following procedure: In a laminar flow hood, rinse with 70% alcohol for 2 min, then rinse with 2.5% sodium hypochlorite for 10 min, and finally rinse with sterile water 4 times. Put them into a pre-sterilized mortar, add a small amount of sterilized quartz sand and an appropriate amount of sterile water, grind thoroughly, let stand for 15 min, pipette 100 μL of the supernatant and spread it on a PDA medium plate, and place it in an incubator at 28 °C for culturing and observation. Randomly pick the growing colonies for purification.

[0035] The disinfection effect on the tissue surface was examined by the rinsing solution test method (Schulz et al., 1993). 0.2 mL of the last rinsing solution was taken and spread on a PDA solid plate for cultivation. The presence or absence of colonies was observed during cultivation. If no colonies appeared, it proved that the material surface was sterilized thoroughly; otherwise, the separation results could not be used.

[0036] The ITS sequence analysis was performed on the isolated and purified strains, and they were identified as Cladosporium cladosporioides, named GLARF-7. The Cladosporium cladosporioides GLARF-7 was isolated from the roots of the rice variety Guangluai 4. Its sequence is as follows: The homology with the accession number KY781386.1 in GenBank reached 99.64%, confirming its taxonomic name as Cladosporium cladosporioides, named Cladosporium cladosporioides GLARF-7. Among them, the ITS sequence fragment of Cladosporium cladosporioides GLARF-7 is shown as SEQ ID NO.1.

[0037] Cladosporium cladosporioides GLARF-7 was cultured on PDA medium at 28 °C for 7 days. The colony was villous, brownish-green, growing flat and circular, with a rough and opaque surface, and dark green on the back. (See Figure 1 A, B).

[0038] (3) Preservation of Cladosporium cladosporioides GLARF-7: The strain was preserved in the Guangdong Microbial Culture Collection Center on December 31, 2024, with the preservation number GDMCC No: 65694.

[0039] Example 2: Inhibitory effect of Cladosporium cladosporioides GLARF-7 on Magnaporthe oryzae

[0040] (1) The preparation of PDA medium was the same as in Example 1;

[0041] (2) Preparation of PA medium: Weigh 2.5 g of lactose, 2.5 g of sucrose, and 1 g of yeast extract and dissolve them in 800 mL of tap water. Then add 20 mL of Prune Juice, adjust the pH to 6.5 with 10 M NaOH, make up the volume to 1000 mL with tap water, add 20 g of agar, heat and dissolve it thoroughly, then dispense it into conical flasks (100 mL of culture solution in each flask), sterilize at 121 °C for 20 min, and store it for later use after cooling.

[0042] (3) Preparation of Cladosporium cladosporioides GLARF-7 and Magnaporthe oryzae discs: Use a puncher to punch holes at the edges of the Cladosporium cladosporioides and Magnaporthe oryzae colonies to obtain circular discs. (Among them, the strain number of the Magnaporthe oryzae is the 70-15 strain).

[0043] (4) For confrontation culture, the medium used was PDA:PA = 1:1. Place the side of the fungal disc with mycelium on the medium, seal it, and after culturing in the dark at 28 °C for 14 d, measure the colony radius and take photos for record.

[0044] Result analysis:

[0045] The endophytic Cladosporium cladosporioides GLARF-7 in rice has an obvious inhibitory effect on the growth of Magnaporthe oryzae. The results are shown in Table 1 and Figure 2 as follows. Compared with the control, the mycelia at the edge of the Magnaporthe oryzae colony in the treatment group were loose, unevenly distributed, and there was an increase in melanin deposition ( Figure 2 A, C), and the colony radius was significantly reduced (Table 1), while the growth of Cladosporium cladosporioides GLARF-7 was not affected. Therefore, it can be seen that Cladosporium cladosporioides GLARF-7 has a significant inhibitory effect on the growth of Magnaporthe oryzae.

[0046] Table 1 Inhibitory effect of Cladosporium cladosporioides GLARF-7 on Magnaporthe oryzae

[0047]

[0048] Note: b1, b2, b3, b4 are the mycelial radii of Magnaporthe oryzae.

[0049] Example 3: Control of Magnaporthe oryzae by the fermentation broth and supernatant of endophytic Cladosporium cladosporioides GLARF-7 in rice roots

[0050] (1) The preparation of PDA medium was the same as in Example 1. For the preparation of liquid PD medium, except without adding agar, the other ingredients were the same as the formula of PDA solid medium. After fully dissolving the weighed medicament, it was dispensed into conical flasks (100 mL of culture solution per flask), stoppered and bandaged, and sterilized at 121 °C for 20 min, and stored for later use after cooling. The preparation of PA medium was the same as in Example 2.

[0051] (2) The preparation of Magnaporthe oryzae fungal discs was the same as in Example 2.

[0052] (3) Preparation of the biological agent of Cladosporium cladosporioides (fermentation broth, supernatant, and sterile filtrate): The Cladosporium cladosporioides fungal discs prepared in step (2) were inoculated into the conical flasks of the liquid PD medium prepared in step (1), and cultured at 30 °C with a shaker speed of 180 rpm for 7 d, and the obtained product was the fermentation broth. Then, it was centrifuged at 10000 rpm for 1 min to obtain the supernatant, and the cells in the supernatant were filtered off with a filter with a pore size of 0.45 μm to obtain the sterile filtrate of Cladosporium cladosporioides.

[0053] (4) For confrontation culture, the medium used was PDA:PA = 1:1. Place the side of the Pyricularia oryzae disc with mycelium on the medium, suck 200 μL of the fermentation broth, supernatant or sterile filtrate and add it to the Oxford cup, seal it, and culture it in the dark at 28 °C for 10 d. Then measure the colony radius of Pyricularia oryzae and take pictures for record.

[0054] Result analysis:

[0055] The results are shown in Table 2 and Figure 3 as follows. Compared with the normally growing Pyricularia oryzae ( Figure 3 A), the fermentation broth ( Figure 3 B) and supernatant ( Figure 3 C) of Cladosporium cladosporioides GLARF-7 endophytic in rice both had obvious inhibitory effects on the growth of Pyricularia oryzae. It could be observed that the mycelia at the edge of the Pyricularia oryzae colony were loose, unevenly distributed, the melanin deposition increased, and the colony radius decreased significantly. Among them, the inhibitory effect of the fermentation broth on Pyricularia oryzae was more significant; the sterile filtrate had no obvious inhibitory effect on the growth of Pyricularia oryzae ( Figure 3 D).

[0056] Table 2 Inhibitory effects of Cladosporium on Pyricularia oryzae of rice

[0057]

[0058]

[0059] Note: b1, b2, b3, b4 are the mycelial radii of Pyricularia oryzae.

[0060] The advantages and positive effects of the technical solution to be protected by the present invention are as follows:

[0061] 1. The Cladosporium cladosporioides GLARF-7 obtained in the embodiment of the present invention is derived from the rice root and can colonize well in the rice tissue. In addition, the antagonistic endophyte screened from rice by the present invention is used for the prevention and control of rice blast. It only shows the increase or decrease of the number of certain microorganisms between specific populations, and does not change the category of the microbial population on rice so as to affect the stability of the population. In addition, due to its long-term coexistence and mutualism with rice, a series of survival mechanisms similar to it have been formed. Spraying it on the surface of rice tissue for disease prevention and control is safer and more reliable for the consumption of rice and its processed products.

[0062] 2. The Cladosporium cladosporioides GLARF-7 in the embodiment of the present invention has a strong inhibitory effect on Pyricularia oryzae, has excellent potential biocontrol effect for preventing and controlling rice blast, and the biological source is environmentally friendly and has little impact on the ecological environment. The Cladosporium cladosporioides obtained by the present invention has low requirements for culture conditions and has good development and application prospects.

[0063] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be covered by the protection scope of the present invention.

Claims

1. An endophytic Cladosporium cladosporioides GLARF-7 of rice, characterized in that: The Cladosporium cladosporioides GLARF-7 was deposited with the Guangdong Microbial Culture Collection Center on December 31, 2024, and the deposit number of the Cladosporium cladosporioides GLARF-7 is GDMCC No: 65694.

2. The endophytic Cladosporium cladosporioides GLARF-7 in rice according to claim 1, characterized in that: The Cladosporium cladosporioides GLARF-7 was isolated and purified from the roots of rice, and the rice variety used for the isolation and purification was Guangluai 4.

3. Use of the endophytic Cladosporium cladosporioides GLARF-7 in claim 1 for controlling rice blast.

4. A biological agent of Cladosporium cladosporioides GLARF-7 endophytic in rice, characterized in that: The biological agent is prepared based on the Cladosporium cladosporioides GLARF-7 described in claim 1 or claim 2.

5. The Cladosporium cladosporioides GLARF-7 biological agent for rice endophyte according to claim 4, characterized in that: The biological agent of the endophytic Cladosporium cladosporioides GLARF-7 is the fermentation broth of Cladosporium cladosporioides GLARF-7 or the supernatant of Cladosporium cladosporioides GLARF-7.

6. The endophytic Cladosporium cladosporioides GLARF-7 biological agent for rice according to claim 5, characterized in that: The preparation method of the fermentation broth of Cladosporium cladosporioides GLARF-7 specifically includes the following steps: inoculating Cladosporium cladosporioides GLARF-7 into a liquid PD medium and culturing it with shaking for 7 days to obtain the fermentation broth of Cladosporium cladosporioides GLARF-7.

7. The Gliocladium ramulosum GLARF-7 biological agent for rice endophyte according to claim 5, characterized in that: The preparation method of the supernatant of Cladosporium cladosporioides GLARF-7 specifically includes the following steps: inoculating Cladosporium cladosporioides GLARF-7 into a liquid PD medium and culturing it with shaking for 7 days, then centrifuging at 10000 rpm for 1 minute and taking the supernatant to obtain the supernatant of Cladosporium cladosporioides GLARF-7.

8. The endophytic Cladosporium cladosporioides GLARF-7 biological agent for rice according to claim 6 or 7, characterized in that: The pH value of the liquid PD medium is 7.

0.

9. The rice endophytic Cladosporium cladosporioides GLARF-7 biological agent according to claim 6 or 7, characterized in that: The liquid PD medium is cultured at 28-30 °C with a shaking rate of 180-200 rpm for 7 days.

10. Use of the biological agent of the endophytic Cladosporium cladosporioides GLARF-7 described in any one of claims 4-9 for controlling rice blast.

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