Rice endophytic penicillium janthinillum WF33R10-2, biological preparation thereof and application thereof in preventing and treating rice blast

Abstract

The invention discloses rice endophytic penicillium janthinillum WF33R10-2, a biological preparation thereof and application of the rice endophytic penicillium janthinillum WF33R10-2 in preventing and treating rice blast, and belongs to the technical field of biopesticides The penicillium janthinillum WF33R10-2 disclosed by the invention is preserved in Guangdong Microbial Culture Collection Center on December 31, 2024, and the preservation number is GDMCC (China General Microbiological Culture Collection Center): 65696; the fermentation liquor or the supernate of the penicillium janthinillum WF33R10-2 is used for preparing a corresponding biological agent; the penicillium janthinense WF33R10-2 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 Penicillium janthinellum WF33R10-2 in rice, its biological agent and application in controlling rice blast disease. Background Art

[0002] Rice (Oryza sativa L.) is one of the most important food crops in the world, with a rather long history of cultivation and consumption. In production, due to the damage of diseases such as rice blast disease, bacterial leaf blight, rice bakanae disease, etc., it seriously affects the high and stable yield of rice. Traditional control of rice blast mainly adopts the method of chemical pesticide treatment, but this method has problems such as large application amount, pesticide residue, and serious environmental pollution. Therefore, we urgently need to adopt natural biological control methods to gradually reduce the use amount of these chemical pesticides. Biological control is a safe, green and new control means. In particular, the research on antagonistic microorganism control in the treatment of rice blast disease is becoming more and more extensive, and the application effect is good. At present, there are many reports on the research of rhizosphere microorganisms in controlling plant diseases, but the research on endophyte biological control is relatively less.

[0003] Endophytic fungi were first discovered in forage grass, and later were also found in woody plants, herbaceous plants, mosses, lichens, seaweeds and other plants. Plant endophytes can live in healthy plants without causing obvious infection symptoms in the host plants. Endophytes can colonize well in plant tissues, co-evolve and symbiotically benefit with the host plants, and the two complement each other. On the one hand, the host plant can create a suitable growth environment for the endophyte; on the other hand, the metabolites and antibacterial active substances produced by the endophyte during its growth process can promote the growth of the host plant and also play a certain role in resisting biotic and abiotic stresses. Redman et al. found that pre-inoculating the non-pathogenic endophytic Colletotrichum magna in melons to watermelon or cucumber seedlings can quickly induce the plant defense response of the seedlings (such as the formation of peroxidase, phenylalanine ammonia-lyase, lignin and salicylic acid). The University of Delhi in India found an endophytic fungus Piriformospora indica, which can promote the growth of corn, tobacco and celery and increase the biomass; when inoculating this fungus to tobacco seedlings during transplantation, its survival rate is more than 22% higher than that of the control. Although Penicillium sp. is a common plant pathogen, it is also a common endophyte in plants. Under natural conditions, the low-abundance Penicillium in the body does not cause disease to the 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, Penicillium sp., as an important natural resource, has various uses and also has the potential to develop biological pesticides.

[0005] At present, there are many reports on the research and utilization of Penicillium sp. as a biocontrol agent, and it has inhibitory effects on various pathogenic fungi such as Rhizoctonia solani, Fusarium oxysporum, and Verticillium dahliae. However, there is less research on its inhibitory effect on Magnaporthe oryzae. Summary of the Invention

[0006] The object of the present invention is to provide an endophytic Penicillium janthinellum WF33R10-2 in rice, its biological agent, and its application in controlling rice blast, aiming to solve the problems of toxicity and residue of existing pesticides for controlling rice blast. The Penicillium janthinellum WF33R10-2 in the present invention is derived from the roots of rice and can colonize well in rice tissues; it has a significant inhibitory effect on Magnaporthe oryzae and has excellent potential biocontrol effects for controlling rice blast; it has low requirements for culture conditions and has good prospects for development and application.

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

[0008] An endophytic Penicillium janthinellum WF33R10-2 in rice, which was deposited at the Guangdong Provincial Microbial Culture Collection Center 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 Penicillium janthinellum WF33R10-2 is GDMCC No: 65696.

[0009] Furthermore, when the endophytic Penicillium janthinellum WF33R10-2 in rice is cultured on PDA medium at 28°C for 7 days, the colony is felt-like, with a flocculent surface, radial folds, white hyphae at the edge, and yellow in the middle.

[0010] Furthermore, the above-mentioned Penicillium janthinellum WF33R10-2 is applied in controlling rice blast.

[0011] Furthermore, the Penicillium janthinellum WF33R10-2 is isolated and purified from the roots of rice, and the rice variety used for isolation and purification is Yuxiangyouzhan.

[0012] Another object of the present invention is to provide a biological agent of endophytic Penicillium janthinellum WF33R10-2 in rice, which is prepared based on the above-mentioned Penicillium janthinellum WF33R10-2.

[0013] Furthermore, the biological agent of endophytic Penicillium janthinellum WF33R10-2 in rice is prepared by liquid fermentation and is the fermentation broth of Penicillium janthinellum WF33R10-2 or the supernatant of Penicillium janthinellum WF33R10-2.

[0014] Furthermore, the preparation method of the fermentation broth of Penicillium janthinellum WF33R10-2 specifically includes the following steps: inoculate Penicillium janthinellum WF33R10-2 into a liquid PD medium and shake culture for 7 days to obtain the fermentation broth of Penicillium janthinellum WF33R10-2.

[0015] Furthermore, the preparation method of the supernatant of Penicillium janthinellum WF33R10-2 specifically includes the following steps: inoculate Penicillium janthinellum WF33R10-2 into a liquid PD medium and shake culture for 7 days, then centrifuge at 10000 rpm for 1 min, take the supernatant to obtain the supernatant of Penicillium janthinellum WF33R10-2.

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

[0017] Furthermore, the specific conditions for the 7-day shake culture are: in the liquid PD medium, culture at 28-30 °C with a shake rate of 180-200 rpm for 7 days.

[0018] Another object of the present invention is to provide the application of the above-mentioned biological agent of endophytic Penicillium janthinellum WF33R10-2 in rice in the prevention and control of rice blast.

[0019] Combined with the above technical solutions and the solved technical problems, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0020] First, Penicillium janthinellum WF33R10-2 obtained in the present invention is derived from rice root tissue and can colonize well in rice tissue; in addition, the antagonistic endophyte screened from rice tissue 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 rice, thus affecting the stability of the population. In addition, because it coexists with rice for a long time and is mutually beneficial and symbiotic, a series of survival mechanisms similar to it are formed. Spraying it on rice tissue for disease prevention and control is safer and more reliable for the consumption of rice and its processed products.

[0021] Second, Penicillium janthinellum WF33R10-2 of the present invention has a strong inhibitory effect on Magnaporthe oryzae, 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. Moreover, Penicillium janthinellum WF33R10-2 obtained in the present invention has low culture conditions and has good development and application prospects.

[0022] Third, the expected benefits and commercial value after the transformation of the technical solution of the present invention are as follows: Based on the microbial resources protected by this patent, which show a significant inhibitory effect on Magnaporthe oryzae in actual production activities, researching and developing bio-based pesticides based on this bacterium for controlling rice blast can effectively reduce the use of chemical pesticides, contribute to the protection of the ecological environment, and ensure food security, with good ecological benefits. Promoting the bacterial agents developed based on this bacterium can reduce the economic losses caused by food production reduction due to the outbreak of rice blast, recover food losses, and bring huge economic benefits. At the same time, the present invention belongs to a green pesticide of microbial origin, conforms to the current trend of pesticide development, and complies with national policy guidance, with great commercial value.

[0023] Fourth, the present invention can solve the technical problem of the single use of microbial pesticides by making different bacterial agents through different treatment methods. Through different treatments, the present invention can be used not only for controlling rice blast but also for stabilizing the microbial community in the rice growth environment and promoting the healthy growth of rice and improving the horizontal disease resistance of rice when used in combination with other microorganisms. 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 Penicillium janthinellum WF33R10-2 provided by the embodiment of the present invention on a 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 Penicillium janthinellum WF33R10-2 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 Penicillium janthinellum WF33R10-2 growing normally, and C is the inhibition diagram of Penicillium janthinellum WF33R10-2 on Magnaporthe oryzae;

[0027] Figure 3 It is an effect diagram of the control of Magnaporthe oryzae by the biological agent of Penicillium janthinellum WF33R10-2 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 Penicillium janthinellum WF33R10-2 on Magnaporthe oryzae, C is the inhibition diagram of the supernatant of Penicillium janthinellum WF33R10-2 on Magnaporthe oryzae; D is the inhibition diagram of the sterile filtrate of Penicillium janthinellum WF33R10-2 on Magnaporthe oryzae. DETAILED DESCRIPTION OF THE INVENTION

[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 of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the scope of protection of the present invention.

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

[0030] Currently, there are problems of pesticide toxicity and residue in the existing pesticides for controlling rice blast. To solve the above technical problems, the present invention provides an endophytic Penicillium janthinellum WF33R10-2 of rice, its biological agent and its application in controlling rice blast.

[0031] Example 1: Isolation, purification and preservation of endophytic Penicillium janthinellum WF33R10-2 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 (20 g of agar needs to be additionally added when making solid PDA medium), fully dissolve and filter with gauze while it is hot, sterilize at 121 °C for 20 min, dispense into petri dishes, cool and store for later use.

[0033] (2) Isolation and purification of endophytic Penicillium in rice:

[0034] The rice sample used for isolation is the cultivated variety "Yuxiangyouzhan". Take the root tissue of this variety growing healthily in the natural rice blast identification nursery, refer to the conventional tissue isolation method and make a slight modification: Wash the healthy root tissue with tap water, dry it, and then perform surface disinfection according to the following procedure: In the ultra-clean workbench, 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 it into a pre-sterilized mortar, add a small amount of sterilized quartz sand and an appropriate amount of sterile water, grind thoroughly, let it stand for 15 min, suck 100 μL of the supernatant and spread it on the PDA medium plate, place it in an incubator at 28 °C for cultivation and observation, and pick and purify the grown colonies at any time.

[0035] The disinfection effect on the tissue surface was examined by the rinsing solution test method (Schulze 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 thoroughly sterilized; otherwise, the separation results could not be used.

[0036] The ITS sequence analysis was performed on the purified strain, and it was identified as Penicillium janthinellum, named Penicillium janthinellum WF33R10-2. The Penicillium janthinellum WF33R10-2 was isolated from the roots of the rice variety "Yuxiangyouzhan", and its sequence is as follows: It has a homology of 97.80% with Penicillium janthinellum with the accession number KM013446.1 in GenBank, confirming its taxonomic name as Penicillium janthinellum, named Penicillium janthinellum WF33R10-2. Among them, the ITS sequence fragment of Penicillium janthinellum WF33R10-2 is shown as SEQ ID NO.1.

[0037] Penicillium janthinellum WF33R10-2 was cultured on PDA medium at 28 °C for 7 days. The colony was felt-like, with a flocculent surface, radial folds, white hyphae at the edge, and yellow in the middle. (See Figure 1 A, B).

[0038] (3) Preservation of Penicillium janthinellum WF33R10-2: The strain was preserved in the Guangdong Provincial Microbial Culture Collection Center on December 31, 2024, with the preservation number GDMCC No: 65696.

[0039] Example 2: Inhibitory effect of Penicillium janthinellum WF33R10-2 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, dissolve them in 800 mL of tap water, 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, fully heat and dissolve, then dispense into conical flasks (100 mL of culture solution per flask), sterilize at 121 °C for 20 min, and store for later use after cooling.

[0042] (3) Preparation of *Penicillium janthinellum* and *Magnaporthe oryzae* discs: Use a borer to punch holes at the edges of the *Magnaporthe oryzae* and *Penicillium* colonies to obtain circular discs. (Among them, the strain number of the rice blast is strain 70-15).

[0043] (4) The medium used for confrontation culture is PDA:PA = 1:1. Place the mycelium-bearing sides of the *Magnaporthe oryzae* and *Penicillium janthinellum* WF33R10-2 discs on the same medium (at a certain distance), 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 *Penicillium janthinellum* WF33R10-2 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 are dense, evenly distributed, and there is an increase in melanin deposition ( Figure 2 A, C), and the colony radius is significantly reduced (Table 1), while the growth of *Penicillium janthinellum* WF33R10-2 is not affected. Therefore, it can be seen that *Penicillium janthinellum* WF33R10-2 has a significant inhibitory effect on the growth of *Magnaporthe oryzae*.

[0046] Table 1 Inhibitory effect of *Penicillium janthinellum* WF33R10-2 on *Magnaporthe oryzae*

[0047]

[0048] Note: b1, b2, b3, b4 are the black boundary radii of *Magnaporthe oryzae*

[0049] Example 3: Control of rice blast by the fermentation broth and supernatant of endophytic *Penicillium janthinellum* WF33R10-2 in rice

[0050] (1) The preparation of the liquid PD medium is the same as in Example 1.

[0051] (2) The preparation of the *Magnaporthe oryzae* and *Penicillium janthinellum* WF33R10-2 discs is the same as in Example 2.

[0052] (3) Preparation of the biological agent (fermentation broth, supernatant, and sterile filtrate) of *Penicillium janthinellum* WF33R10-2: Inoculate the *Penicillium janthinellum* WF33R10-2 disc prepared in step (2) into the PD culture solution conical flask prepared in step (1), place it at 28 °C, and culture it on a shaker at a rate of 180 rpm for 7 d. The obtained solution is the fermentation broth. Then centrifuge at 10000 rpm for 1 min to obtain the supernatant, and filter the supernatant through a bacterial filter with a pore size of 0.45 μm to remove the bacteria in the supernatant to obtain the sterile filtrate of *Penicillium janthinellum* WF33R10-2.

[0053] (4) For confrontation culture, the medium used was PDA:PA = 1:1. The side of the Pyricularia oryzae disc with mycelium was placed on the medium. 200 μL of the fermentation broth, supernatant, or sterile filtrate was added to the Oxford cup, sealed, and cultured in the dark at 28 °C for 10 d. Then, the colony radius was measured and photographed.

[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 the endophytic Penicillium janthinellum WF33R10-2 in rice roots 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. It could be seen that the fermentation broth and supernatant of Penicillium janthinellum WF33R10-2 significantly inhibited the growth of Pyricularia oryzae, and the sterile filtrate had no obvious effect on the growth of Pyricularia oryzae ( Figure 3 D), indicating that the biological agents (fermentation broth and supernatant) of Penicillium janthinellum WF33R10-2 had obvious potential control effects on rice blast.

[0056] Table 2 Inhibitory effects of the biological agent of Penicillium janthinellum WF33R10-2 on Pyricularia oryzae

[0057]

[0058]

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

[0060] Based on the detection results of the embodiments of the present invention, the advantages and positive effects of the technical solution to be protected by the present invention are as follows:

[0061] First, the obtained Penicillium janthinellum WF33R10-2 of the present invention is derived from the rice root tissue and can colonize well in the rice tissue. In addition, the antagonistic endophyte screened from the rice tissue by the present invention is used for the control of rice blast, which only shows the increase or decrease of the number of a certain microorganism among 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 mutualistic symbiosis with rice, a series of survival mechanisms similar to it have been formed. Spraying it on the rice tissue for disease control is safer and more reliable for the consumption of rice and its processed products.

[0062] Second, the Penicillium janthinellum WF33R10-2 of the present invention has a strong inhibitory effect on Magnaporthe oryzae, has excellent potential biocontrol effects on preventing and controlling rice blast, and is environmentally friendly in terms of biological sources and has little impact on the ecological environment. Moreover, the Penicillium janthinellum WF33R10-2 obtained in 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 modifications, equivalent replacements, and improvements 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 Penicillium janthinellum WF33R10-2 of rice, characterized in that: The Penicillium janthinellum WF33R10-2 was deposited at the Guangdong Microbial Culture Collection Center on December 31, 2024, and the deposit number of the Penicillium janthinellum WF33R10-2 is GDMCC No: 65696.

2. The endophytic Penicillium janthinellum WF33R10-2 in rice according to claim 1, characterized in that: The Penicillium janthinellum WF33R10-2 was isolated and purified from the roots of rice, and the rice variety used for the isolation and purification is Yuxiangyouzhan.

3. Application of the endophytic Penicillium janthinellum WF33R10-2 in rice as claimed in claim 1 in the prevention and control of rice blast.

4. A biological agent of endophytic Penicillium janthinellum WF33R10-2 in rice, characterized in that: The biological agent is prepared based on the Penicillium janthinellum WF33R10-2 as claimed in claim 1 or claim 2.

5. The endophytic Penicillium janthinellum WF33R10-2 biological agent according to claim 4, characterized in that: Prepared by liquid fermentation, the fermentation broth of Penicillium janthinellum WF33R10-2 or the supernatant of Penicillium janthinellum WF33R10-2.

6. The endophytic Penicillium janthinellum WF33R10-2 biological agent according to claim 5, characterized in that: The preparation method of the fermentation broth of Penicillium janthinellum WF33R10-2 specifically comprises the following steps: inoculating Penicillium janthinellum WF33R10-2 into a liquid PD medium and culturing it with shaking for 7 days to obtain the fermentation broth of Penicillium janthinellum WF33R10-2.

7. The endophytic Penicillium janthinellum WF33R10-2 biological agent according to claim 5, characterized in that: The preparation method of the supernatant of Penicillium janthinellum WF33R10-2 specifically comprises the following steps: inoculating Penicillium janthinellum WF33R10-2 into a liquid PD medium and culturing it with shaking for 7 days, then centrifuging at 10000 rpm for 1 minute, taking the supernatant to obtain the supernatant of Penicillium janthinellum WF33R10-2.

8. The endophytic Penicillium janthinellum WF33R10-2 biological agent for rice according to claim 6 or claim 7, characterized in that: The pH value of the liquid PD medium is 7.

0.

9. The endophytic Penicillium janthinellum WF33R10-2 biological agent according to claim 6 or claim 7, characterized in that: The specific conditions for culturing with shaking for 7 days are: culturing the liquid PD culture medium at 28-30 °C with a shaking rate of 180-200 rpm for 7 days.

10. Application of the endophytic Penicillium janthinellum WF33R10-2 biological agent as claimed in any one of claims 4-9 in the prevention and control of rice blast.

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