CN111471601A - Method for culturing brown spot germs of sugarcane - Google Patents
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Abstract
The Cercospora cubensis is inoculated to a culture medium, the temperature is set to be 5-35 ℃, and the pH value of the culture medium is 4-10, the culture medium is one of PDA, PSA, MEA, CAM, Czapek, L BA, N6, MS and WA, the optimum temperature of the culture method of the Cercospora cubensis is 25 ℃, the optimum pH value of hypha growth is 5, the optimum culture mediums are PDA, PSA and YPD, fructose, beef extract and yeast powder are most beneficial to hypha growth of germs and are the optimum carbon and nitrogen sources of the Cercospora cubensis, and the influence of the culture conditions and the nutritional conditions on the growth of the Cercospora cubensis is determined, so that a foundation is laid for revealing the occurrence and epidemic rules of the Cercospora cubensis.
Description
Technical Field
The invention belongs to the technical field of microorganisms, relates to a separation culture method of sugarcane brown spot pathogenic bacteria, and particularly relates to a culture method of sugarcane brown spot pathogenic bacteria Cercospora longipes.
Background
Sugarcane (Saccharum spp.) is the most important sugar crop in China, and diseases are important factors influencing sugarcane production. At present, 130 sugarcane diseases are reported in the world, and more than 50 sugarcane infectious diseases are recorded in China. Among them, leaf diseases such as brown rust (brown rust), tip rot (pokkah boeng), brown strip disease (brown strip), brown spot (brown spot) and the like caused by fungi are common in sugarcane areas in China, and pose serious threats to sugarcane production.
Brown spot disease in sugarcane caused by Cercospora longipes e.e. butler was first discovered in india in 1906 and is now widely distributed in sugarcane production areas worldwide. The brown spot mainly damages sugarcane leaves, the damaged leaves show yellow specks at the early stage of disease incidence, then the long fusiform red brown spots with yellow halos are enlarged to form, when the field humidity is high, gray mildew layers are generated on the back surfaces of the leaves, the leaves are withered in advance due to serious disease incidence, and the leaves are burnt, so that the sugarcane production is seriously lost.
Brown spot is one of main leaf diseases of sugarcane, and early researchers carry out a large amount of investigation and analysis on the distribution and damage conditions of the disease in different sugarcane areas in China, and find that the disease is widely distributed in Guangxi, Yunnan, Guangdong, Guizhou, Hainan and the like in sugarcane areas in China, is damaged all the year round and has certain influence on the normal growth of the sugarcane. At present, the prevention and control of the sugarcane brown spot still mainly adopt chemical agents, and the evaluation of the prevention and control effects of a plurality of chemical agents on the disease shows that the azoxystrobin benzoate has good prevention and control effects on the sugarcane brown spot. Understanding the biological characteristics of the germs is the basis for deeply understanding the disease occurrence rule, and further provides a theoretical basis for effective prevention and control of diseases. However, related reports on the biological characteristics of pathogenic bacteria of the saccharomycete are lacked in the existing research, and the research on the culture environment of the saccharomycete is still blank.
Disclosure of Invention
The occurrence rule and the effective prevention and control theory of the cercospora canescens caused by the cercospora canescens are not deep enough, a foundation is laid for revealing the occurrence and prevalence rule of the cercospora canescens, and the biological characteristics of the cercospora canescens are required to be known. Based on the above, the invention provides a method for culturing the brown spot pathogen of sugarcane, which probes the influence of culture conditions and nutritional conditions on the growth of hyphae of cercospora saccharum.
In order to achieve the above object, the present invention adopts the following technical solutions.
A method for culturing sugarcane brown spot germs, wherein the sugarcane brown spot germs are Cercospora saccharina (Cercospora longipes) capable of causing sugarcane brown spot disease, the Cercospora saccharina is inoculated into a culture medium, and the culture conditions are set to be 5-35 ℃ and the pH value of the culture medium is 4-10.
In a preferred embodiment of the culture method of the present invention, the carbon source of the culture medium is selected from one of glucose, galactose, fructose, mannose, soluble starch, maltose, sorbitol, inositol, and sucrose. The influence of different carbon sources on the hypha growth of the cercospora saccharensis is researched, so that the cercospora saccharensis can utilize various monosaccharides, polysaccharides and soluble starch as the carbon sources. Further preferably, the carbon source of the medium is selected from one of fructose, glucose, maltose and inositol. Among the various carbon sources tested, fructose, glucose, maltose and inositol were used as carbon sources to facilitate the growth of the cercospora saccharina hyphae.
As a preferred embodiment of the cultivation method according to the invention, the nitrogen source of the medium is selected from the group consisting of beef extract, yeast powder, peptone, glycine, NH4Cl、NH4NO3、Ca(NO3)2、NaNO3One kind of (1). The influence of different nitrogen sources on the growth of the hyphae of the cercospora saccharensis is researched, so that the cercospora saccharensis can utilize an inorganic nitrogen source and an organic nitrogen source to different degrees, but the organic nitrogen source is more beneficial to the growth of the hyphae of the cercospora saccharensis. Further preferably, the beef extract, the yeast powder and the peptone screened by the method are organic nitrogen sources suitable for culturing the cercospora sacchari.
The present inventors have found that cercospora saccharensis can grow on all 10 media tested, but the difference in growth is large, and further preferably, the medium is selected from one of PDA, PSA, YPD, CAM and MEA.
In a preferred embodiment of the culture method of the present invention, the temperature of the cercospora saccharensis inoculated in the PDA culture medium is 10 to 30 ℃ and the pH of the PDA culture medium is 4 to 10. According to the research of the invention, the cercospora saccharum is inoculated in a PDA culture medium and can grow under the condition that the culture temperature is 10-30 ℃; has wide application range to pH, and is especially favorable to acid-biased growth environment.
As a preferred embodiment of the culture method, PDA or PSA culture medium is selected to inoculate and culture the cercospora saccharensis, the culture temperature is 25 ℃, and the pH of the PDA or PSA culture medium is 5. The culture conditions and the nutrient conditions have obvious influence on the growth of the cercospora crassa hyphae, the temperature of 25 ℃ and the pH of 5 are the optimal temperature and the pH for the growth of germs, PDA and PSA are the optimal culture medium, and the optimal carbon and nitrogen sources are fructose, beef extract and yeast powder.
The culture method of the brown spot pathogen of the sugarcane at least has the following beneficial effects or advantages.
1) The invention takes Cercospora longipes as a research object, and researches the influence of temperature, pH, culture medium types, different carbon sources and nitrogen sources on the growth of hyphae of the Cercospora longipes by measuring the growth of bacterial colonies. The temperature range of the hypha growth of the cercospora saccharensis is 5-35 ℃, and the optimal temperature is 25 ℃; the optimal pH value for hypha growth is 5, and the hypha can grow at the pH value of 4-10. The cercospora sacchar grew on all 10 media tested, and the PDA and PSA media favoured the hyphal growth of this strain, whereas the MS and N6 media were not suitable for growth of this strain. The comparison of the influence of different carbon and nitrogen sources on the growth of hyphae shows that the fructose, the beef extract and the yeast powder are most beneficial to the growth of hyphae of pathogenic bacteria and are the best carbon and nitrogen sources of the pathogenic bacteria.
2) The invention defines the influence of culture conditions and nutritional conditions on the growth of hyphae of cercospora saccharum, and lays a foundation for disclosing the occurrence and prevalence rules of cercospora leaf spot of sugarcane. The culture method of the sugarcane brown spot germs established by the invention enriches the research on the culture environment of the sugarcane tail spores and is beneficial to disclosing the prevalence rule of the sugarcane brown spot disease caused by the sugarcane tail spores.
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FIG. 1 shows the results of measurements of the growth of Cercospora saccharensis hyphae at different temperatures.
FIG. 2, results of measurements of growth of cercospora sacchar filaments at different pH values.
Detailed Description
In order to facilitate understanding of the objects, technical solutions and effects of the present invention, the present invention will be further described in detail with reference to examples.
Collecting sugarcane brown spot disease sample and separating pathogenic bacteria
The brown spot disease samples of sugarcane are collected in the Dongsanzake sugarcane production base in the official town of Wenyuan county, Guangdong province. Collected brown spot disease leaves are placed in an ice box and brought back to a laboratory, the shape of pathogenic bacteria is observed by microscopic examination with an optical microscope, and the pathogenic bacteria are separated and cultured by adopting a conventional tissue separation method. The separated cercospora saccharensis is propagated and stored on a potato glucose agar medium (PDA) plate for later use.
Field hazard of brown spot of sugarcane
Brown spot of sugarcane only harms sugarcane leaves. Half spots can be generated on the front and back of the leaf, the early stage is red oval scab, and the later stage is reddish brown with yellow halo and irregular.
Morphology of cercospora saccharensis
The shape of conidia of Cercospora longipes (Cercospora longipes) pathogen is observed by a microscope, wherein conidia peduncles are clustered and brown, have knee bending folding points and multiple diaphragms, and the conidia are inverted stick-shaped, colorless and have multiple diaphragms with the size of 35.0-120.0 × 4.0.0-7.0 mm.
Colony morphology of brown spot pathogen of sugarcane on PDA culture medium: the hyphae are compact, but the colony grows slowly, the diameter is about 10-15 mm after 3 weeks of culture, the surface is gray to light black, and red pigment is contained.
Identification of Cercospora saccharensis
According to the morphological observation and the research result of the culture shape of the brown spot pathogen of the sugarcane, refer to the monograph: guo Ying lan, Liu Xi \29710, 2005, Chinese Eugenia: twenty-fourth volume: cercospora records, determined that sugarcane brown spot disease is caused by sugarcane tail spores (Cercospora longipes).
Effect of temperature on hyphal growth
A bacterial cake with the diameter of 5mm is taken from the edge of a colony of the Cercospora longipes (Cercospora longipes) cultured for 20d and is transferred to the center of a PDA (personal digital assistant) plate culture medium by using a sterilization hole puncher, the bacterial cake is respectively placed at 5, 10, 15, 20, 25, 30, 35 and 40 ℃ for culture, the diameter of the colony is measured by a cross method every 7d, and the morphology of the colony is observed and recorded. Each treatment was repeated 3 times.
The results of the tests on the hyphal growth of Cercospora longipes (Cercospora longipes) at different temperatures are shown in fig. 1. As shown in FIG. 1, the isolates can grow at a temperature of 5-35 ℃; when the culture temperature is not higher than 10 ℃, hyphae grow slowly, and the diameter of a colony cultured for 35d is not more than 10 mm; in the temperature range of 15-30 ℃, hyphae grow rapidly, the diameter of a colony cultured for 35d grows to 15-25 mm, the optimal growth temperature is 25 ℃, and the diameter of the colony reaches 23.4 mm; hyphae can not grow at the culture temperature of higher than 35 ℃.
Effect of pH on hyphal growth
The pH value of PDA culture medium is adjusted to 4, 5, 6, 7, 8, 9 and 10 by using 0.1 mol/L HCI and 0.1 mol/L NaOH, the germ cake is inoculated in the center of PDA culture medium plates with different pH values according to the method, the PDA culture medium plates are cultured in an incubator at 25 ℃ under constant temperature and in dark, the diameter of the colony is measured by adopting a cross method every 7d, the colony morphology is observed and recorded, and each treatment is repeated for 3 times.
The results of measurements of the growth of cercospora sacchar filaments at different pH are shown in FIG. 2. As shown in FIG. 2, the isolate shows a wide adaptation range to pH, is relatively suitable for a meta-acid growth environment, and can grow under 7 pH gradient conditions of 4-10; the diameter of the colonies cultured for 35d all grow over 10mm, wherein the diameter of the colonies under the condition of pH5 is the largest and reaches 24.9 mm; the isolate also grew well on plates of more alkaline PDA medium, with a colony diameter of 12.1mm at pH 10.
Effect of different media on hyphal growth
PDA, potato 200 g/L, glucose 20 g/L, agar powder 15 g/L;
PSA, potato 200 g/L, cane sugar 20 g/L, agar powder 15 g/L;
MEA, 30 g/L of malt extract powder, 3.0 g/L of soybean peptone and 15 g/L of agar powder;
CAM comprises corn extract powder 2.0 g/L and agar powder 15 g/L;
Czapek：NaNO33g/L、KH2PO41g/L、MgSO4·7H2O 0.5g/L、KCl 0.5g/L、FeSO40.01 g/L, 30 g/L of sucrose and 15 g/L of agar powder;
l BA, NaCl 10 g/L, peptone 10 g/L, yeast powder 5 g/L and agar powder 15 g/L;
YPD, glucose 20 g/L, peptone 20 g/L, yeast powder 10 g/L and agar powder 15 g/L;
N6：KNO32830mg/L、(NH4)2SO4463mg/L、CaCl2·2H2O 166mg/L、MgSO4·7H2O185mg/L、KH2PO4400mg/L、FeSO4·7H2O 27.8mg/L、MnSO4·4H2O 4.4mg/L、ZnSO4·7H2O1.6mg/L、H2BO30.8 mg/L, KI 1.6 mg/L, glycine 2 mg/L and nicotinic acid 0.5 mg/L;
MS：KNO31900mg/L、(NH4)2SO41650mg/L、KH2PO4170mg/L、MgSO4·7H2O 370mg/L、CaCl2·2H2O 440mg/L、KI 0.83mg/L、H2BO36.2mg/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O8.6mg/L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.25mg/L、CoCl2·6H2O 0.025mg/L、Na2EDTA 37.25mg/L、FeSO4·7H2o27.85 mg/L, inositol 100 mg/L, glycine 2 mg/L, nicotinic acid 0.5 mg/L, pyridoxine hydrochloride 0.5 mg/L, thiamine hydrochloride 0.1 mg/L, sucrose 30 g/L, and agar powder 15 g/L;
WA agar 15 g/L.
N6 and MS in the culture are purchased from Sigma company, and other culture media are prepared according to a formula; the prepared culture medium is sterilized in a sterilization pot at 121 ℃ under moist heat and high pressure for 20min for later use.
The bacterial cake WAs transferred to the center of a plate made of 10 different media, such as PDA, PSA, MEA, CAM, Czapek, L BA, YPD, N6, MS and WA, using the above-mentioned method.
The growth of Cercospora saccharina (Cercospora longipes) in different cultures is shown in Table 1. As seen from table 1, cercospora sacchar (c. longipes) can grow on all 10 media tested, but the growth was greatly different. From the aspects of colony morphology and growth speed, PDA, PSA and YPD are most suitable for hyphal growth of the saccharomycete, and are CAM and MEA; hyphae were extremely sparse on WA, whereas MS and N6 media were not suitable for hyphal growth of C.saccharum.
Table 1 effect of different media on mycelial growth of sugar cane urospora (c
Note: different lower case letters after the same column of data indicate significant differences (P < 0.05).
Effect of different carbon sources on hyphal growth
Czapek culture medium is used as a base, and glucose, galactose, fructose, mannose, soluble starch, maltose, sorbitol and inositol in equal amounts are respectively used for replacing sucrose in the Czapek culture medium to prepare culture media with different carbon sources. Inoculation of the pathogens, culturing, observation of colony growth and measurement of colony diameter were performed as described. Each treatment was repeated 3 times.
The effect of different carbon sources on the hyphal growth of c.longipes is shown in table 2. As seen from table 2, the sugar cane urospora (c. longipes) can utilize various monosaccharides, polysaccharides and soluble starch as carbon sources, and different carbon sources have certain influence on the growth of hyphae thereof. Of the 8 carbon sources tested, fructose is the best carbon source for cercospora sacchar (c. longipes), the second best of glucose, maltose and inositol, and is poorly utilized for soluble starch.
Table 2 effect of different carbon sources on hyphal growth of sugar cane urospora (c
Note: different lower case letters after the same column of data indicate significant differences (P < 0.05).
Effect of different Nitrogen sources on hyphal growth
Taking Czapek culture medium as a base, and respectively taking equal amounts of beef extract, yeast powder, peptone, glycine and NH4Cl、NH4NO3And Ca (NO)3)2Replacing NaNO therein3Different nitrogen source culture media are prepared. Inoculation of the pathogens, culturing, observation of colony growth and measurement of colony diameter were performed as described. Each treatment was repeated 3 times. The effect of different nitrogen sources on the hyphal growth of c.longipes is shown in table 3.
Table 3 effect of different nitrogen sources on hyphal growth of sugar cane urospora (c
Note: different lower case letters after the same column of data indicate significant differences (P < 0.05).
Growth of cercospora saccharea (c. longipes) hyphae in different nitrogen source media is shown (table 3): sugar cane urosporium (c. longipes) can utilize inorganic and organic nitrogen sources to varying degrees, but different nitrogen sources have significant effects on the growth of pathogenic hyphae. The most suitable nitrogen sources in the test cercospora saccharea (c. longipes) were beef extract and yeast powder, followed by peptone, vs NH4Cl and NH4NO3Is poorly utilized.
In conclusion, the culture conditions and the nutritional conditions have obvious influence on the growth of hyphae of the sugarcane oxysporum (C.longipes), the hyphae can grow at 5-35 ℃ and at the pH value of 4.0-10, the optimum growth temperature is 25 ℃, the pH value is 5, PDA and PSA are the optimum culture media, and fructose, beef extract and yeast powder are the optimum carbon and nitrogen sources.
The present invention has been further described with reference to the examples, but the present invention is not limited to the above-described embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.
Claims (10)
1. The method for culturing the sugarcane brown spot germs is characterized in that the sugarcane brown spot germs are sugarcane Cercospora longipes (Cercospora longipes) capable of causing sugarcane brown spot disease, the sugarcane Cercospora longipes is inoculated into a culture medium, and the culture conditions are set to be 5-35 ℃ and 4-10 of the pH value of the culture medium.
2. The culture method according to claim 1, wherein the carbon source of the medium is one selected from the group consisting of glucose, galactose, fructose, mannose, soluble starch, maltose, sorbitol, inositol, and sucrose.
3. The culture method according to claim 1, wherein the nitrogen source of the medium is selected from the group consisting of beef extract, yeast powder, peptone, glycine, NH4Cl、NH4NO3、Ca(NO3)2、NaNO3One kind of (1).
4. The culture method according to claim 1, wherein the medium is one selected from the group consisting of PDA, PSA, MEA, CAM, Czapek, L BA, YPD, N6, MS and WA.
5. The method according to claim 1, wherein the temperature for culturing the cercospora saccharensis inoculated in the PDA culture medium is 10 to 30 ℃ and the pH of the PDA culture medium is 4 to 10.
6. The culture method according to claim 4, wherein the medium is one selected from the group consisting of PDA, PSA, YPD, CAM and MEA.
7. The culture method according to claim 6, wherein the medium is one selected from the group consisting of PDA, PSA, and YPD.
8. The culture method according to claim 2, wherein the carbon source of the medium is one selected from fructose, glucose, maltose and inositol.
9. The culture method according to claim 3, wherein the nitrogen source of the medium is one selected from the group consisting of beef extract, yeast powder, and peptone.
10. The culture method according to claim 1, wherein the cercospora saccharensis is inoculated and cultured in PDA or PSA medium at 25 ℃ and pH 5.
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Application publication date: 20200731 |