A culture medium and method for inducing mass sporulation of gaeumannomyces graminis

Abstract

The present application is suitable for the technical field of plant pathogenic microorganism, and provides a culture medium and method for inducing large sporulation of wheat take-all pathogen, which comprises the following steps: after an isolated Gaeumannomyces graminis trittici special strain is activated, the strain is inoculated into the culture medium for inducing large sporulation of the wheat take-all pathogen to conduct sporulation culture; after mycelium covers the plate, the aerial mycelium is gently wiped off with sterilized absorbent cotton balls, and then the plate is placed in a 25-28 DEG C near-ultraviolet lamp incubator to culture for 4 days. By comparing sporulation effects of different culture media under the same temperature, illumination and humidity conditions, the present application is significantly better than other culture media. The present application effectively solves the problems that the Gaeumannomyces graminis trittici special strain does not sporulate or has low sporulation under conventional culture conditions, and greatly improves sporulation efficiency. By using the method, uniform and stable characteristics of conidia can be obtained, and sufficient fungus source guarantee is provided for subsequent strain identification, pathogenic mechanism research and disease-resistant variety screening.

Description

Technical Field

[0001] This invention relates to the field of plant pathogenic microbial technology, specifically a culture medium and method for inducing massive spore production by wheat blast fungus. Background Technology

[0002] Wheat blast is a devastating fungal disease caused by *Magnaporthe oryzae* pathotype *Triticum* (MoT), a dangerous and devastating fungal disease in tropical and subtropical wheat-producing regions. It was first discovered in Paraná, Brazil in 1985 and subsequently spread rapidly to major wheat-producing areas in Brazil and to South American countries such as Bolivia, Paraguay, and Argentina. In recent years, wheat blast has entered South Asia (Bangladesh, 2016) and Africa (Zambia, 2018) through international wheat trade, posing a serious threat to global wheat production. The pathogen can infect the wheat spike, leaves, leaf sheaths, glumes, awns, stem nodes, and seeds, with spike infection being the most typical, manifesting as partial or complete whitening of the spike, leading to total crop failure in severe cases. Under suitable conditions, yield loss can reach 100%. In Bangladesh, during the first outbreak in 2016, approximately 15,000 hectares of wheat were affected, with an average yield loss of 25-30%. In Bolivia, the first outbreak in 1996 resulted in 80% yield loss, and the following year caused 100% crop failure. Wheat blast fungus belongs to the Ascomycota phylum, and its host range includes wheat, barley, triticale, and various grass weeds. Currently, the main production-available resistance resources rely on the 2NS chromosome segment, derived from *Aegilops ventricosa*, which has been widely used in CIMMYT and other breeding programs. However, highly virulent strains that overcome 2NS resistance (such as 16MoT001) have emerged in South America, indicating a loss of resistance. Therefore, clarifying the pathogenic variations of wheat blast fungus, conducting screening for resistant varieties, and studying the resistance mechanisms have become core tasks for the current control of this disease. However, the prerequisite and foundation for all this research is obtaining a large number of consistent, active conidia.

[0003] Currently, sporulation of wheat blast fungus mainly relies on traditional methods such as PDA or OMA media. While these methods can achieve sporulation of wheat blast fungus to some extent, they still have many shortcomings: the sporulation rate is unstable, with some strains producing almost no sporulation; the culture period is long, requiring several weeks or even longer to induce a small amount of sporulation; and the sporulation capacity varies greatly among strains from different sources, affecting the reproducibility of subsequent pathogenicity assays and resistance screening.

[0004] Therefore, in view of the above situation, there is an urgent need to provide a culture medium and method for inducing massive sporulation of wheat blast fungus, so as to overcome the shortcomings in current practical applications. Summary of the Invention

[0005] The purpose of this invention is to provide a culture medium and method for inducing massive sporulation of wheat blast fungus, thereby addressing the problems mentioned in the background art.

[0006] The present invention is achieved by providing a culture medium for inducing massive spore production of wheat blast fungus, wherein the culture medium is a wheat bran medium containing wheat bran powder and agar.

[0007] As a further aspect of the present invention: the wheat bran powder is cooked wheat bran powder, and the culture medium formula is: 40 g / L cooked wheat bran powder and 20 g / L agar.

[0008] As a further aspect of the present invention, the culture medium is further supplemented with ampicillin and streptomycin sulfate, wherein the final concentration of both ampicillin and streptomycin sulfate is 10 ng / mL.

[0009] The present invention also provides a method for inducing massive sporulation of wheat blast fungus, comprising the following steps:

[0010] S1. Activate the specialized strain of *Pyrrosia lingua* on an activation medium;

[0011] S2. Prepare the above-mentioned wheat bran culture medium, pour the sterilized sporulation culture medium into a petri dish, and obtain wheat bran culture medium plates.

[0012] S3. Take the mycelial block of the strain activated in step S1, inoculate it on the wheat bran culture medium plate prepared in step S2, and incubate it in the dark at 25℃ for 7-10 days until the mycelium covers the plate.

[0013] S4. Remove the aerial mycelium from the surface of the plate, air dry, and then place the plate upright in a 25-28℃ environment under near-ultraviolet light for 4 days to induce sporulation.

[0014] S5. Add sterile water to the plate after inducing sporulation, collect the bacterial solution containing spores, and prepare a spore suspension.

[0015] As a further aspect of the present invention: the activation medium in step S1 is PDA medium, and the activation culture conditions are dark culture at 25℃ for 5-7 days.

[0016] As a further aspect of the present invention: the light conditions for dark culture in step S3 are 16 h / 8 h light-dark alternating culture.

[0017] As a further aspect of the present invention: the sterilization conditions in step S2 are 121°C high-pressure steam sterilization for 20 minutes;

[0018] The method of adding ampicillin and streptomycin sulfate to the sporulation medium plates is to add them at a volume ratio of 1:1000 after sterilization.

[0019] As a further aspect of the present invention: the method for removing aerial hyphae in step S4 is to gently wipe them off with a sterile defatted cotton ball without damaging the agar medium.

[0020] The drying time is 30-40 minutes.

[0021] As a further aspect of the present invention: the wavelength of the near-ultraviolet lamp in step S4 is 365 nm and the power is 18 W.

[0022] As a further aspect of the present invention: the method for preparing the spore suspension in step S5 is specifically as follows:

[0023] Add 5 mL of sterile water to the plate, gently scrape the plate to elute the spores, collect the eluent, add another 5 mL of sterile water to wash once more, combine the two eluents, vortex disperse for 2-3 min, filter with a magic filter cloth, centrifuge at 5000 rpm for 10 min, discard the supernatant, add 1 mL of sterile water to resuspend the precipitate, and obtain the spore suspension.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] The method of this invention can induce stable conidia and produce a large number of conidia, providing important practical experience for the development of conidial induction technology for other *Pyrrosia* fungi. Furthermore, the operation of this invention is feasible, greatly improving conidial production efficiency, effectively saving manpower, material resources, and a great deal of valuable time for researchers, accelerating the research process related to wheat blast, and has great application value.

[0026] This invention provides a rapid method for inducing sporulation of wheat blast fungus. The sporulation conditions are clearly defined, and the sporulation conditions, including culture temperature, light, substrate, and humidity, have been optimized. The optimal temperature range, light duration, solid culture medium, and constant humidity for stable sporulation of the strain have been screened. These conditions are simple, controllable, and highly reproducible, and can be applied on a large scale to the construction of standardized laboratory inoculation systems. The culture medium is high-quality and low-cost. Compared with commonly used media such as PDA and OMA, wheat bran, which is widely available and inexpensive, is selected as the optimal sporulation substrate, significantly reducing experimental costs. Furthermore, inefficient and easily contaminated media are eliminated during the screening process, further improving sporulation stability. The results are accurate and reliable. Biological replicates and blank control groups were set up for sporulation experiments to eliminate interference from environmental factors, contaminants, and operational errors. The data on spore yield, activity, and morphology are objective and reliable, with minimal experimental error. The sporulation test results are authentic, reliable, and highly reproducible. It has high reference value in the industry, improves the artificial sporulation induction technology system of wheat blast fungus, fills the shortcomings of existing technology, and can provide standardized technical reference for the study of the biological characteristics of wheat blast fungus, the screening of wheat disease-resistant germplasm, and the research on disease control. It has outstanding value for promotion and scientific research. Attached Figure Description

[0027] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 Colony morphology of *Pyrrosia lingua* strain on different culture media; from left to right: OMA medium, PDA medium, and wheat bran medium (MF medium).

[0029] Figure 2 The morphology of conidia of the wheat-specific strain of *Pyrhodotorula gravidii* on wheat bran medium (MF medium).

[0030] Figure 3 To compare the spore concentrations obtained by diluting the *Pyrrosia lingua* strain with 1 mL of sterile water after induction with near-ultraviolet light for 4 days on different sporulation media, sporulation was performed on PDA medium, OMA medium, and wheat bran medium (MF medium). Detailed Implementation

[0031] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] The present invention will be further explained below with reference to specific embodiments.

[0033] The present invention provides a culture medium for inducing massive sporulation of wheat blast fungus, wherein the culture medium is wheat bran medium (hereinafter referred to as MF medium), which contains 40 g / L cooked wheat bran powder and 20 g / L agar;

[0034] The culture medium also contains ampicillin and streptomycin sulfate, with a final concentration of 100 μg / mL for both ampicillin and streptomycin sulfate.

[0035] This invention also provides a method for inducing massive sporulation of wheat blast fungus, comprising the following steps:

[0036] S1. The isolated wheat-specific strain of *Pyrrosia lingua* was activated in PDA medium.

[0037] S2. Prepare the above-mentioned MF medium, autoclave it at 121℃ for 20 min, pour it into a petri dish, and add ampicillin and streptomycin sulfate (final concentration 100 μg / mL) at a volume ratio of 1:1000 to obtain MF medium plates.

[0038] S3. Take fresh mycelia from the edge of the colony of the activated strain and place them on the above-mentioned MF medium plates. Incubate in the dark at 25-28℃ for 7-10 days until the mycelia cover the plate. The activation method is to incubate in MF medium at 25℃ for 16 h / 8 h with alternating light and dark conditions.

[0039] S4. Gently wipe away the aerial hyphae with a sterile defatted cotton ball, being careful not to break the agar medium. After drying in a clean bench, after 30-40 minutes, place the plate upright in an incubator at 25-28℃ with a 365 nm, 18 W near-ultraviolet lamp and incubate for 4 days.

[0040] S5. Gently scrape the plate, rinse twice with 5 ml of sterile water, collect the spore-containing solution in a 50 ml centrifuge tube, vortex for 2-3 min, filter through a filter cloth, centrifuge the filtrate at 5000 rpm for 10 min, discard the supernatant, add 1 ml of sterile water to resuspend the filtrate, and transfer it to a collection tube to obtain a spore suspension. The spore concentration is measured using a hemocytometer. Through these steps, rapid and large-scale induction of sporulation of wheat blast fungus and preparation of spore suspension are achieved.

[0041] In this embodiment, the method is highly operable, simple to operate, and easy to control the culture conditions. It solves the problems of non-sporogenesis of wheat special types or low sporogenesis yield of existing methods, greatly improves sporogenesis efficiency, and can obtain characteristic and stable conidia, so as to facilitate the identification of wheat special types, morphological variation and pathogenic mechanism research, and screening of disease-resistant varieties.

[0042] Please see Figures 1-3 To provide a clear and complete description of the technical solution of the present invention, the embodiments of the present invention also provide the following implementation examples. Unless otherwise specified, the experimental methods in the following implementation examples are conventional methods. Unless otherwise specified, the experimental materials in the following embodiments are all purchased from conventional biochemical reagent stores.

[0043] I. Strains and Culture Media

[0044] The strain is wheat blast fungus YN001.

[0045] The three sporulation media and formulations of wheat blast fungus used are: PDA medium, OMA medium and MF medium (Table 1).

[0046] Table 1. Sporulation medium formulation for wheat blast fungus

[0047] culture medium formula PDA medium 200 g potatoes, 20 g glucose, 18 g / L agar, and diluted with pure water to a final volume of 1 L. OMA medium Boil 30 g of rolled oats in pure water for 1 hour, filter through four layers of cheesecloth to remove sediment, add 100 ml of freshly squeezed tomato juice and 15 g of agar, and bring the volume to 1 L with pure water. The pH is 7.2. MF medium 40 g / L cooked wheat bran powder, 20 g / L agar

[0048] The average concentrations of spore suspensions on the three wheat blast culture media provided by this invention are shown in Table 2.

[0049] Table 2. Average concentration of spore suspensions of wheat blast fungus on different sporulation media.

[0050] culture medium Average concentration of spores in suspension (spores / mL) PDA medium <![CDATA[(2.00±0.82)×10 4 ]]> OMA medium <![CDATA[(4.67±0.91)×10 4 ]]> MF medium <![CDATA[(52.00±10.97)×10 4 ]]>

[0051] II. Inoculation and Culture of Wheat Blight

[0052] Each culture medium was inoculated with mycelial blocks of the same size as wheat blast fungus. All treatment groups were incubated in a 25°C incubator for 7–10 days with alternating light and dark settings of 16 h / 8 h. After approximately 10 days of incubation, the mycelium was scraped off, and the mixture was then placed in a 25–28°C incubator under near-ultraviolet light to produce sporulations, with three replicates per group.

[0053] III. Data Collection and Analysis

[0054] The growth state of mycelia covering the plate in the culture medium was recorded by camera. The number of wheat blast fungus spores in each culture dish was recorded under a microscope by hemocytometer. The spore concentration per unit area was calculated as a measure.

[0055] Because the spore concentration data of each group contained a large number of zero values ​​and extremely high values, did not conform to a normal distribution, and had unequal variances between groups, the Kruskal-Wallis H nonparametric test was used to analyze the overall differences between groups, and the Tukey HSD test was used to complete multiple comparisons.

[0056] IV. Effects of different culture medium formulations on the growth of wheat blast fungus

[0057] After 10 days of cultivation, observations revealed that different culture media significantly affected the growth of wheat blast fungus. In traditional sporulation media, the mycelium on OMA medium did not completely cover the plate, resulting in sparse and soft mycelial growth; on PDA medium, the mycelium growth was sparse, with the smallest colony coverage and the worst growth-promoting effect. In contrast, the MF medium provided by this invention exhibited the best growth-promoting effect, with mycelium completely covering the plate, and dense, vigorous colonies, significantly superior to OMA and PDA media. Figure 1 ).

[0058] V. Effects of different culture medium formulations on sporulation of wheat blast fungus

[0059] Under uniform conditions of 4 days of UV-induced culture, the sporulation yields of different culture medium formulations showed significant differences. Among the conventional media, OMA medium exhibited better sporulation levels than PDA, with an average spore concentration of (4.67±0.91)×10⁻⁶.4 The concentration of spores per mL was only sufficient to meet the basic requirements for conidial differentiation of the strain; the sporulation capacity of PDA medium was the weakest, at only (2.00±0.82)×10⁻⁶. 4 The concentration of spores / mL indicates that the nutrient ratio of this culture medium is more suitable for mycelial growth and not for the large-scale induction of wheat blast fungus spores (Table 2 and ). Figure 3 ).

[0060] Compared to two traditional culture media, the MF-specific culture medium designed in this invention has a highly significant promoting effect on sporulation of wheat blast fungus, with an average sporulation yield of (52.00±10.97)×10⁻⁶. 4 The sporulation rate was increased by about 10 times compared to OMA medium, and the total sporulation was an order of magnitude higher.

[0061] The original sporulation data for each parallel sample are detailed in Table 3. Because the sporulation concentration data for each group contained numerous zero values ​​and extremely high values, did not conform to a normal distribution, and had unequal variances between groups, the Kruskal-Wallis H nonparametric test was used for overall inter-group difference analysis. GraphPad Prism software was used to perform pairwise comparisons of sporulation yields between the three culture media (Dunn test for multiple comparisons). The Dunn multiple comparison results (Table 4) showed that the sporulation yield of the pathogen on wheat bran medium (MF) was significantly higher than that on OMA medium (P=0.0003) and PDA medium (P<0.0001), while there was no statistically significant difference in sporulation yield between OMA and PDA media (P=0.4833). These statistical results further confirm the significant advantage of the wheat bran medium of this invention compared to traditional culture media in inducing massive sporulation of wheat blast pathogens.

[0062] Table 3 Spore suspension concentration

[0063] culture medium repeat <![CDATA[Spore suspension concentration (×10 4 )]]> MF1 1 20 MF1 2 5 MF1 3 65 MF1 4 20 MF1 5 15 MF2 1 30 MF2 2 55 MF2 3 145 MF2 4 30 MF2 5 20 MF3 1 35 MF3 2 85 MF3 3 125 MF3 4 35 MF3 5 95 PDA1 1 0 PDA1 2 0 PDA1 3 0 PDA1 4 5 PDA1 5 0 PDA2 1 0 PDA2 2 5 PDA2 3 5 PDA2 4 0 PDA2 5 10 PDA3 1 0 PDA3 2 5 PDA3 3 0 PDA3 4 0 PDA3 5 0 OMA1 1 10 OMA1 2 10 OMA1 3 5 OMA1 4 5 OMA1 5 0 OMA2 1 5 OMA2 2 5 OMA2 3 5 OMA2 4 10 OMA2 5 5 OMA3 1 0 OMA3 2 0 OMA3 3 0 OMA3 4 5 OMA3 5 5

[0064] Group comparison Mean rank difference Z value Corrected P-value Significance PDA vs OMA −6.533 1.401 0.4833 ns PDA vs MF −24.77 5.312 ＜0.0001 **** OMA vs MF −18.23 3.911 0.0003 ***

[0065] Table 4 Dunn Multiple Comparisons

[0066] Note: PDA is potato dextrose agar medium, OMA is oat agar medium, and MF is wheat bran medium.

[0067] The growth state of mycelia covering the plate in the culture medium was recorded by camera. The number of wheat blast fungus spores in each culture dish was recorded under a microscope by hemocytometer. The spore concentration per unit area was calculated as a measure.

[0068] Considering both total spore production and practical value, traditional OMA and PDA media are insufficient for efficient spore preparation of wheat blast fungus. Repeated propagation can easily lead to strain viability decay and amplified experimental errors. MF media, through formula optimization, achieves a significant increase in spore production, yielding a high-concentration spore suspension in a single culture. This greatly simplifies pathogenicity testing, drug toxicity screening, and artificial inoculation, making it a high-quality culture medium formula suitable for large-scale spore preparation of wheat blast fungus. Furthermore, standardized inoculation and stable culture environments can reduce intragroup variation, further improving the standardized use of this culture medium.

[0069] In summary, this invention proposes a highly efficient sporulation culture medium, wheat bran medium, which achieves efficient sporulation of wheat blast fungus without increasing the difficulty of acquisition or economic costs. This facilitates the collection of wheat blast fungus spore suspensions for further scientific research.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for inducing massive sporulation of wheat blast fungus, characterized in that, The culture medium is a wheat bran medium, which contains wheat bran powder and agar.

2. The culture medium according to claim 1, characterized in that, The wheat bran powder is cooked wheat bran powder, and the culture medium formula is: 40 g / L cooked wheat bran powder and 20 g / L agar.

3. The culture medium according to claim 1, characterized in that, The culture medium also contains ampicillin and streptomycin sulfate, with a final concentration of 100 μg / mL for both ampicillin and streptomycin sulfate.

4. A method for inducing massive sporulation of wheat blast fungus, characterized in that, Includes the following steps: S1. Activate the specialized strain of *Pyrrosia lingua* on an activation medium; S2. Prepare the wheat bran culture medium according to any one of claims 1 to 3 by pouring the sterilized sporulation culture medium into a petri dish to obtain a wheat bran culture medium plate. S3. Take the mycelial block of the strain activated in step S1, inoculate it on the wheat bran culture medium plate prepared in step S2, and incubate it in the dark at 25℃ for 7-10 days until the mycelium covers the plate. S4. Remove the aerial mycelium from the surface of the plate, air dry, and then place the plate upright in a 25-28℃ environment under near-ultraviolet light for 4 days to induce sporulation. S5. Add sterile water to the plate after inducing sporulation, collect the bacterial solution containing spores, and prepare a spore suspension.

5. The method according to claim 4, characterized in that, The activation medium mentioned in step S1 is PDA medium, and the activation culture conditions are dark culture at 25℃ for 5~7 days.

6. The method according to claim 4, characterized in that, The light conditions for dark culture in step S3 are 16 h / 8 h light and dark alternating culture.

7. The method according to claim 4, characterized in that, The sterilization conditions described in step S2 are: high-pressure steam sterilization at 121°C for 20 minutes; The method of adding ampicillin and streptomycin sulfate to the sporulation medium plates is to add them at a volume ratio of 1:1000 after sterilization.

8. The method according to claim 4, characterized in that, The method for removing aerial hyphae in step S4 is to gently wipe them off with a sterile defatted cotton ball without breaking the agar medium. The drying time is 30-40 minutes.

9. The method according to claim 4, characterized in that, The near-ultraviolet lamp mentioned in step S4 has a wavelength of 365 nm and a power of 18 W.

10. The method according to claim 4, characterized in that, The specific method for preparing the spore suspension in step S5 is as follows: Add 5 mL of sterile water to the plate, gently scrape the plate to elute the spores, collect the eluent, add another 5 mL of sterile water to wash once more, combine the two eluents, vortex disperse for 2-3 min, filter with a magic filter cloth, centrifuge at 5000 rpm for 10 min, discard the supernatant, add 1 mL of sterile water to resuspend the precipitate, and obtain the spore suspension.

Images