CN112029666A - Citrus limosum spore-forming culture method and application thereof - Google Patents
Citrus limosum spore-forming culture method and application thereof Download PDFInfo
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- 244000052616 bacterial pathogen Species 0.000 claims abstract description 28
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- 238000005286 illumination Methods 0.000 claims abstract description 15
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a citrus brown spot germ spore-forming culture method and application thereof. The culture time is researched by taking Alternaria tenuissima as an object; the culture temperature; low-temperature induction; illumination; a carbon source; a nitrogen source; the influence of the culture medium type on the hypha growth and the spore yield of Alternaria tenuissima is realized, and then the spore production condition is optimized by combining an orthogonal Latin square test. The optimal spore production condition after the optimization of the pathogenic bacteria is 25 ℃, under the condition of full-black and dark culture, the optimized PSY is used as a culture medium (12.0g of maltose replaces 2.0g of sucrose in the PSY culture medium, 20.0g of potassium nitrate replaces 10.0g of peptone and 5.0g of yeast extract), the ultraviolet irradiation is carried out on the pathogenic bacteria for 20min and 15 ℃ induction is carried out for 3h every day in a specific time period, and the spore production is measured after the culture is carried out for 4 days.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a citrus brown spot germ spore-forming culture method and application thereof.
Background
Citrus (Citrus reticulata Blanco) is a rutaceae plant and is a generic name of oranges, tangerines, oranges, kumquats, pomelos, poncirus trifoliata and the like. The orange pulp is sour, sweet and delicious, is deeply favored by men, women, the old and the young, and is the second largest fruit which is second to apples in China.
Citrus is susceptible to diseases and insect pests, and there are dozens of common diseases, such as Citrus yellow shoot (Candidatus Liberibacter asiaticus), Citrus scab (Citrus scab sphaerica fawcetti), Citrus anthracnose (Colletotrichum gloeosporoides), and so on.
The citrus brown patch can cause a great amount of fallen leaves and fallen fruits of citrus, even withered branches and tips, and can have great influence on the yield and quality of citrus. The citrus brown spot is discovered in australia for the first time in 1903, and in recent years, the citrus brown spot occurs in China in Guangdong, Guangxi, Chongqing and the like in succession, and the harm is gradually increased. However, the citrus brown spot belongs to new diseases in China, and no systematic and complete research is carried out on pathogens of the citrus brown spot, and a complete and reliable control method is not available.
The citrus brown spot pathogen is Alternaria tenuissima (Alternaria tenuissima), and the citrus hosts thereof comprise: snow pomelo, sha mandarin orange, fragrant citrus fruit and grapefruit. However, the spore yield of the pathogenic bacteria is relatively low, and certain difficulty is formed in indoor research, so that the experiment performs biological characteristic research and spore yield optimization experiment on alternaria tenuissima to provide certain basis for preventing and treating citrus brown spot.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for culturing citrus brown spot germ to produce spores and application thereof, and aims to solve part of problems in the prior art or at least alleviate part of problems in the prior art.
The invention is realized in such a way that the method for culturing the citrus brown spot germ to produce spores comprises the following steps: maltose and potassium nitrate; the culture temperature is 25 ℃; the illumination condition is 24h dark + (10-20) min ultraviolet irradiation, and the culture is carried out under the condition of low-temperature induction at 15 ℃ for 2-3 h.
Further, the amount of potassium nitrate was 20.0 g/L.
Further, maltose was used in an amount of 12.0 g/L.
Further, the light condition was 24h dark +20min uv irradiation.
Further, the induction condition is low temperature induction at 15 ℃ for 3 h.
Further, the spore production culture time was 4 days.
Further, the pathogen is alternaria tenuissima.
The method for culturing the citrus brown spot germ with the spores is applied to the culture of the citrus brown spot germ with the spores.
In summary, the advantages and positive effects of the invention are:
this test was carried out on Alternaria tenuissima (Alternaria tenuissima) and the culture time was investigated; the culture temperature; low-temperature induction; illumination; a carbon source; a nitrogen source; influence of culture medium type on hypha growth and sporulation amount of Alternaria tenuissima (A. tenuissima), and optimization of sporulation conditions by combining an orthogonal Latin square test. The research result shows that: the 4 th day is the optimum culture time for pathogenic bacteria to produce spores. The optimum temperature for pathogenic bacteria spore production is 15 ℃. The optimal induction temperature for the hypha growth of pathogenic bacteria is 20 ℃, and the optimal induction temperature for spore production is 15 ℃. The most suitable illumination condition for pathogenic bacteria spore production is 24h dark +10 minUV. The influence difference of different carbon sources and nitrogen sources on the hypha growth and the spore yield of pathogenic bacteria is obvious, the most suitable carbon source for the hypha growth is starch, and the most suitable carbon source and the nitrogen source for spore production are respectively maltose and potassium nitrate. The different culture media have obvious influence difference on the hypha growth and spore production of pathogenic bacteria, and the optimal culture media for the hypha growth and the spore production are PSA and PSY respectively. The optimal sporulation conditions after the pathogenic bacteria are optimized are that 12.0g/L maltose is used as a carbon source, 20.0g/L potassium nitrate is used as a nitrogen source, the illumination condition is 24h dark +20min UV, and the induction is carried out at the low temperature of 15 ℃ for 3 h.
Through range analysis and variance analysis, the optimal sporulation conditions of Alternaria tenuissima are determined to be 25 ℃, under the condition of full-black and dark culture, the optimized PSY is used as a culture medium (12.0g/L maltose replaces 2.0g/L sucrose, 20.0g/L potassium nitrate replaces 10.0g/L peptone and 5.0g/L yeast extract), the ultraviolet irradiation is carried out on the pathogenic bacteria for 20min and 15 ℃ induction is carried out for 3h every day in a specific period, and the sporulation amount is measured after the culture is carried out until the 4 th day. The visible ultraviolet radiation and low-temperature treatment can promote the sporulation of pathogenic bacteria.
The experiment adopts orthogonal Latin square design, so thatGet the original requirement 34The test of the group can be completed by only 9 groups, which greatly reduces the workload of the test.
Drawings
FIG. 1 shows the effect of cultivation time on the amount of produced spores of Alternaria gracilis.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.
For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.
The normal temperature in the following embodiments of the present invention refers to a natural room temperature condition in four seasons, and is not subjected to additional cooling or heating treatment, and is generally controlled at 10 to 30 ℃, preferably 15 to 25 ℃.
The invention discloses a method for culturing citrus brown spot germ to produce spores and application thereof, and the specific embodiment is shown in the following examples.
The media involved in the present application are as follows:
(1) potato dextrose agar medium (PDA).
(2) Potato sucrose agar medium (PSA).
(3) Potato carrot medium (PCA).
(4) Cza medium (CZA).
(5) Oat groats medium (OMA).
(6) Carrot agar medium.
The above culture media are treated by wet heat sterilization for 30min according to plant pathological experiment technology.
(7) Sucrose yeast extract medium (PSY): 2.0g of cane sugar, 5.0g of yeast extract, 10.0g of peptone, 17.0g of agar and 1000ml of water, and the pH value is natural.
(8) Corn meal agar medium: 15.0g of agar, 17.0g of corn flour and 1000ml of water, and the pH value is natural.
(9) Cantino PYG medium (PYG): 17.0g of agar, 1.25g of yeast extract, 1.25g of peptone, 3.0g of glucose and 1000ml of water, and the pH value is natural.
(10) Tomato juice agar medium (TA): 125.0g of tomato, 15.0g of agar and 1000ml of water, and the pH value is adjusted to 7.
(11) Carbon source basal medium: 0.5g of potassium chloride, 0.5g of magnesium sulfate heptahydrate, 3.0g of sodium nitrate, 0.001g of ferrous sulfate, 1.0g of dipotassium hydrogen phosphate, 20.0g of agar and 1000ml of water, and the pH value is natural.
(12) Nitrogen source basal medium: 0.5g of potassium chloride, 1.0g of dipotassium phosphate, 0.001g of ferrous sulfate, 0.5g of magnesium sulfate heptahydrate, 20.0g of cane sugar, 20.0g of agar and 1000ml of water, and the pH value is natural.
Examples
The causative bacterium of cercospora citrifolia referred to in this application is Alternaria tenuissima (Alternaria tenuissima).
The detection method involved in the present application:
colony diameter: two diameters of the colonies were measured by a cross method, and the average was taken as measurement data.
And (3) measuring the sporulation amount: 1ml of sterile water was added to each dish, pathogenic spores were sufficiently washed out, and the number of spores was measured using a hemocytometer.
The data analysis method comprises the following steps: the single-factor test results in 2.3 were processed using the Duncan new repolarization method of software SPSS 17.0, and the test results in 2.4 were analyzed using a software orthogonal design assistant.
1. Influence of culture time on growth and spore yield of alternaria tenuissima
Inoculating 5mm fungus cake into PDA culture medium, and culturing at constant temperature at 25 deg.C for 24 hr in dark. Samples were taken at 1, 2, 3, 4, 5, 6 and 7d after incubation, and sporulation was performed as described above, with 3 replicates per treatment set.
The results are shown in FIG. 1 and show that Alternaria gracilis produces different spore yields at different cultivation times. The largest spore yield of Alternaria tenuissima reaches 4.63 multiplied by 10 when the culture time is day 45The number of the seeds per dish is the most unfavorable for the sporulation of pathogenic bacteria on day 1, and the sporulation amount is only 0.38 multiplied by 105One cell/dish.
2. Influence of culture temperature on spore yield of Alternaria gracilis
Inoculating 5mm of the fungus cake into PDA culture medium, and culturing in incubator at 5 deg.C, 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C and 35 deg.C for 24 hr in dark. Each process set 3 sets of repetitions. After 4d of culture, the spore production was determined as described above.
The results are shown in Table 1 below. As is clear from Table 1, the culture temperature significantly affected the spore yield of the strain. When the culture temperature is 15 ℃, the sporulation yield is the maximum and reaches 17.4 multiplied by 105The spore yield is the lowest and is only 0.08 multiplied by 10 when the culture temperature is 35 ℃ per dish5One cell/dish.
TABLE 1 Effect of culture temperature on the spore yield of Alternaria gracilis
Table1 Effect of culture temperature on the sporulation of A.tenuissima
Note: the SSR method is adopted for inspection, and the data in the table are mean values +/-standard deviation. The letters are identical indicating that the difference is not statistically significant (P > 0.05).
3. Influence of low-temperature induction on growth and spore yield of alternaria tenuissima
Inoculating 5mm of fungus cake into PDA culture medium, culturing in dark at 25 deg.C, culturing at 5 deg.C, 10 deg.C, 15 deg.C and 20 deg.C for 2 hr every day, and inducing at low temperature. Each process set 3 sets of repetitions. After 4d of culture, the colony diameter and spore yield were measured as described above.
The results are shown in Table 2 below, and it is clear from Table 2 that the effect of low-temperature induction on the hyphal growth rate and spore yield of pathogenic bacteria is significant. The growth rate of pathogenic bacteria hyphae is the fastest when the pathogenic bacteria hyphae are induced for 2 hours at 20 ℃, the diameter of the hyphae reaches 48.1mm, the growth rate of the pathogenic bacteria hyphae is the slowest when the pathogenic bacteria hyphae are induced for 2 hours at 10 ℃, and the diameter of the hyphae is only 43.0 mm. When the induction temperature is 15 ℃, the spore production of pathogenic bacteria is most favorable, and the spore production amount reaches 6.23 multiplied by 105The spore yield is only 3.02 multiplied by 10 when the strain per dish and the induction temperature is 5 ℃ which is most unfavorable for the spore production of pathogenic bacteria5One cell/dish.
TABLE 2 Effect of Low temperature Induction on the growth and sporulation of Alternaria tenuissima
Table2 Effect of low temperature induction on the mycelial growth and sporulation of A.tenuissima
Note: the SSR method is adopted for inspection, and the data in the table are mean values +/-standard deviation. The letters are identical indicating that the difference is not statistically significant (P > 0.05).
4. Influence of illumination on growth and spore yield of alternaria tenuissima
Inoculating 5mm fungus cake into PDA culture medium, and culturing at room temperature (25-28 deg.C) under the conditions of 24h illumination, 24h illumination +10min ultraviolet, 24h dark +10min ultraviolet, 12h illumination and 12h dark alternation +10min ultraviolet. Each process set 3 sets of repetitions. After 4d of culture, the colony diameter and spore yield were measured as described above.
As shown in Table 3, it is understood from Table 3 that the pathogenic bacteria produced the largest amount of spores under the illumination conditions of 24h dark +10min UV, and the amount of spores produced reached 11.22X 105The illumination condition of one dish is 24h illumination, the illumination condition is not suitable for sporulation, and the sporulation quantity is only 0.03 multiplied by 105One cell/dish.
TABLE 3 Effect of light on the amount of produced spores of Alternaria gracilis
Table3 Effect of light treatments on the sporulation of A.tenuissima
Note: the SSR method is adopted for inspection, and the data in the table are mean values +/-standard deviation. The letters are identical indicating that the difference is not statistically significant (P > 0.05).
5. Influence of carbon source on growth and spore yield of alternaria tenuissima
Respectively inoculating 5mm fungus cakes into carbon source basal mediums which are prepared by replacing sucrose with equal mass of mannitol, sucrose, glucose, maltose, starch, sorbitol, xylose, chitin and lactose, and culturing at 25 ℃ for 24h in the dark by using the carbon source basal mediums as a control group. Each process set 3 sets of repetitions. After 4d of culture, the colony diameter and spore yield were measured as described above.
The results are shown in Table 4. As can be seen from Table 4, there are significant differences in the growth rate of hyphae and the amount of sporulation of pathogenic bacteria on different carbon-source media. The carbon source with the highest growth speed of the alternaria tenuissima filaments is starch, the diameter of a bacterial colony reaches 29.5mm, the lowest growth speed is xylose, and the diameter of the hypha is only 23.6 mm. Pathogenic bacteria produce the largest amount of spores on a culture medium using maltose as a carbon source, and the spore production amount is 1.11 multiplied by 104The spore yield is minimum and none is produced on a culture medium taking CK, lactose, chitin, xylose, glucose and mannitol as carbon sourcesAnd (4) carrying out sporulation.
TABLE 4 influence of carbon sources on the growth and sporulation yield of Alternaria tenuissima
Table4 Effect of carbon sources on the mycelial growth and sporulation of A.tenuissima
Note: the SSR method is adopted for inspection, and the data in the table are mean values +/-standard deviation. The letters are identical indicating that the difference is not statistically significant (P > 0.05).
6. Influence of nitrogen source on growth and spore yield of alternaria tenuissima
Respectively inoculating 5mm of the fungus cakes into a nitrogen source basal medium which replaces sodium nitrate with equal mass of sodium nitrite, hydrolyzed milk protein, beef extract, ammonium nitrate, peptone, glycine, potassium nitrate and urea, and culturing the fungus cakes under the conditions of 25 ℃ and 24h in the dark by taking the nitrogen source basal medium as a control group. Each process set 3 sets of repetitions. After 4d of culture, the colony diameter and spore yield were measured as described above.
The results are shown in Table 5. As is clear from Table 5, the amount of sporulation of pathogenic bacteria cultured using potassium nitrate as a nitrogen source was 4.72X 10, which is the maximum4Compared with the control group, the difference of the other nitrogen sources is not obvious, and the spore production is not facilitated. The culture medium using urea and glycine as nitrogen sources and the control group have the worst spore production amount, and no spore is produced.
TABLE 5 influence of Nitrogen sources on the growth and sporulation yield of Alternaria gracilis
Table5 Effect of nitrogen sources on the mycelial growth and sporulation of A.tenuissima
Note: the SSR method is adopted for inspection, and the data in the table are mean values +/-standard deviation. The letters are identical indicating that the difference is not statistically significant (P > 0.05).
7. Influence of culture medium on growth and spore yield of alternaria tenuissima
Inoculating 5mm of the bacterial cake into PDA, PSA, PSY, PYG, PCA, CZA, TA, OMA culture medium, carrot agar culture medium, and corn agar culture medium, and culturing at 25 deg.C for 24 hr in the dark. Each process set 3 sets of repetitions. After 4d of culture, the colony diameter and spore yield were measured as described above.
The results are shown in Table 6. As is clear from Table 6, different media affected both the growth of hyphae and the spore yield of pathogenic bacteria. Pathogenic bacteria have the fastest hypha growth speed on PSA, the hypha diameter is 45.2mm, and the hypha growth speed on TA is the slowest, and the hypha diameter is only 30.7 mm. The most favorable culture medium for producing the spores of Alternaria gracilis is PSY, and the spore yield reaches 95.00 multiplied by 104The culture medium which is most unfavorable for spore production is corn meal agar culture medium, and the spore production amount is only 1.40 multiplied by 104One cell/dish.
TABLE 6 Effect of the Medium on the growth and sporulation of Alternaria tenuissima
Table6 Effect of medium on the mycelial growth and sporulation of A.tenuissima
Note: the SSR method is adopted for inspection, and the data in the table are mean values +/-standard deviation. The letters are identical indicating that the difference is not statistically significant (P > 0.05).
8. Multifactor experiment for optimizing sporulation conditions
According to the result of the single-factor sporulation amount, the maltose dosage and the KNO are respectively taken3The dosage, the 15 ℃ induction time under the 25 ℃ culture condition and the ultraviolet lamp induction time under the 24h dark condition are 4 factors in total, and L is established9(34) Orthogonal table, 3 sets of repetitions per treatment setup. And after obtaining a better combination through range analysis, judging the influence degree of each factor on the alternaria citrifolia spore yield through variance analysis, and finally determining the culture condition most suitable for alternaria citrifolia spore yield.
The factor bit levels are shown in table 7 below.
TABLE 7 factor bit-level test for spore yield condition optimization
Table1 Factors and levels of orthogonal test for optimization of sporulation condition
Note: maltose and KNO in table3The usage amount is based on PSY culture medium, sucrose, peptone and yeast extract in the culture medium are respectively replaced, and the usage amount of water is 1000 ml.
The results of the experiments are shown in tables 8 and 9, and it is evident from the results of the range analysis in table 8 that the theoretically best combination of levels is A1B3C3D 3. The influence degree of each factor on the spore yield of Alternaria tenuissima is ultraviolet treatment time, maltose addition amount, potassium nitrate addition amount and 15 ℃ induction time from high to low. As can be seen from Table 9, the influence of 4 factors on spore productivity did not differ significantly.
TABLE 8 orthogonal test results and range analysis of the influence of different factors on the number of spores produced by Alternaria gracilis
Table8 Orthogonal test results and range analysis of the effects of different factors on the number of sporulation of A.tenuissima
Note: k represents the average sporulation amount of each factor at each level; r represents the difference in average spore yield (maximum average spore yield-minimum average spore yield) for each level of the same factor.
TABLE 9 analysis of variance of results of orthogonal tests on the influence of different factors on the spore yield of Alternaria gracilis
Table9 Variance analysis of orthogonal test for influence of different factors on sporulation of pathogen of A.tenuissima
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principles of the present invention are intended to be included within the scope of the present invention.
Claims (8)
1. The method for producing spores of citrus brown spot germs is characterized in that the components of a culture medium comprise: maltose and potassium nitrate; the culture temperature is 25 ℃; the illumination condition is 24h dark + (10-20) min ultraviolet irradiation, and the culture is carried out under the condition of low-temperature induction at 15 ℃ for 2-3 h.
2. The method for producing spores of citrus brown spot germ according to claim 1, wherein: the amount of potassium nitrate is 20.0 g/L.
3. The method for producing spores of citrus brown spot germ according to claim 1, wherein: the amount of maltose used is 12.0 g/L.
4. The method for producing spores of citrus brown spot germ according to claim 1, wherein: the illumination condition is 24h dark +20min ultraviolet irradiation.
5. The method for producing spores of citrus brown spot germ according to claim 1, wherein: the induction condition is low temperature induction at 15 ℃ for 3 h.
6. The method for producing spores of citrus brown spot germ according to claim 1, wherein: the spore production culture time is 4 days.
7. The method for producing spores of citrus brown spot germ according to claim 1, wherein: the pathogenic bacteria are Alternaria tenuissima.
8. Use of a method according to any one of claims 1 to 7 for the sporulation culture of citrus limosa.
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