A method for rapid and large-scale induction of spore production of sugarcane red rot pathogen Colletotrichum fusae

Abstract

The invention provides a method for rapidly and massively inducing spore production of sugarcane red rot pathogen Fusarium sphaerocephala, and relates to the technical field of microorganisms. The method for rapidly and massively inducing spore production of sugarcane red rot pathogen Fusarium sphaerocephala mainly comprises the following steps: activating the isolated Fusarium sphaerocephala strain, carrying out spore production culture in a Yunnan pine solid spore production medium, and then inoculating the strain on an OMA solid medium for culture. The spore production amount can reach 5.5×10 5 / cm 2 ~1×10 7 / cm 2 The present invention overcomes the shortcomings of the prior art. The method is highly operable, simple to operate, and the culture conditions are easy to control. It solves the problem that the sickle thorn spore does not produce spores or the existing method produces a small amount of spores, greatly improves the spore production efficiency, and can obtain conidia with stable characteristics, so as to facilitate the identification of sickle thorn spores, morphological variation research, pathogenic mechanism research and disease-resistant variety screening.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a method for rapidly and massively inducing spore production of sugarcane red rot pathogen Colletotrichum fusae. Background Art

[0002] Sugarcane is one of the important economic crops in tropical and subtropical regions, one of the most important sugar crops in the world, one of the important sources of sugar, and an important raw material for light industry, chemical industry and energy. Sugarcane red rot caused by Colletotrichumfalcatum Went is a global fungal disease. So far, 68 countries have reported the disease, which is often called the "cancer" of sugarcane. At present, the disease is widespread and can occur and cause harm throughout the growth period of sugarcane. The red rot bacteria infect newly planted sugarcane and perennial sugarcane, resulting in sugarcane seedling failure, lack of ponds and broken ridges, resulting in sugarcane yield reduction, and shortening the perennial years. The most destructive stage is when the pathogen infects the sugarcane stem, causing the sugarcane stem to turn red, rot, and the sugarcane plant to wither. The pathogen secretes sucrose invertase, which reduces the purity of sugarcane juice and causes a loss of more than 30.8% of sucrose content. Generally, the yield is reduced by 29.1%. The yield reduction in severely affected sugarcane areas can reach 100%. It is an important fungal disease that seriously affects sugarcane yield and variety.

[0003] The mutation rate of Fusarium spp. is fast, and the emergence of new subspecies leads to the "loss" of disease resistance in sugarcane varieties. The morphological variation, pathogenic variation, pathogenic mechanism research of Fusarium spp. and the corresponding screening of disease-resistant varieties and research on disease resistance mechanisms have been hot topics for many years and have attracted widespread attention. Induced conidia is a key link in classification and identification and morphological variation research, and it is also the premise of pathogenic variation research, and the basis for screening disease-resistant varieties and research on sugarcane disease resistance mechanisms. How to quickly and easily obtain a large number of conidia largely determines the progress and success of the research. However, at present, the isolated and cultured Fusarium spp. produces less or no conidia during the artificial cultivation process, which seriously hinders the research progress of Fusarium spp. pathogenic mechanism and identification of disease-resistant varieties.

[0004] Breeding and planting disease-resistant varieties are the most economical and effective prevention and control measures for sugarcane red rot. Clarifying the variation of Fusarium spp. and identifying and planting disease-resistant varieties for different pathogenic species are the most commonly used and effective prevention and control measures abroad. Therefore, a large number of conidia is an important prerequisite and basis for pathogenic variation research and disease-resistant variety screening. At present, the main method for producing conidia of Fusarium spp. is to use oat culture medium to obtain it. However, this method has some disadvantages: (1) low conidia production; (2) long culture cycle, only a small amount of spores will be produced after a long period of culture; (3) there are still many strains that do not produce conidia or the conidia production does not reach the inoculation amount.

[0005] Therefore, seeking a culture method for Fusarium sphaerocephala that is easy to operate and produces a large number of spores is of great significance for the identification of Fusarium sphaerocephala, the study of morphological variation, the study of pathogenic mechanism, the screening of disease-resistant varieties and the study of disease resistance mechanism. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a method for rapidly and massively inducing spore production of the sugarcane red rot fungus Fusarium spp. The method has strong operability, simple operation, and easy control of culture conditions. It solves the problems that Fusarium spp. does not produce spores or the existing methods produce little spores, greatly improves the spore production efficiency, and can obtain conidia with stable characteristics, so as to facilitate the identification of Fusarium spp., the study of morphological variation, the study of pathogenic mechanism, and the screening of disease-resistant varieties.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solutions:

[0008] A method for rapidly and massively inducing spore production of sugarcane red rot fungus Colletotrichum fusae, the method comprising the following steps:

[0009] S1. placing the isolated Fusarium sphaeroides strain in a culture medium for activation;

[0010] S2. Prepare a solid spore-forming medium for Yunnan pine: set it up according to the following formula: KH2PO4 1.0 g / L, KNO3 1.0 g / L, MgSO4·7H2O 1.0 g / L, KCl 0.5 g / L, glucose 0.5 g / L, sucrose 0.2 g / L, agar 18 g / L, add water to make up to 1 L; then sterilize it with high-pressure steam at 121°C for 25 min and pour it into a culture dish. After the culture medium solidifies, put 5-10 sterilized old leaves of Yunnan pine in each culture dish to prepare a solid spore-forming medium for Yunnan pine;

[0011] S3, taking fresh mycelium from the edge of the colony of the activated strain and placing it on the solid spore-forming medium of Yunnan pine for cultivation until sickle-shaped conidia are produced in the mycelium blocks near the pine needles under a microscope;

[0012] S4. Take the mycelial block where conidia are produced on the solid conidial medium of Yunnan pine and place it in OMA solid medium and culture it in the dark at 28° C. to achieve rapid and large-scale induction of conidia production of sugarcane red rot fungus Fusarium sphaerocephala.

[0013] Preferably, the method for isolating and obtaining the Fusarium sphaeroides strain comprises the following steps:

[0014] S1-1. Select the diseased midrib, stem or seed of sugarcane as raw materials, cut small pieces of 3mm×5mm at the junction of diseased and healthy sugarcane with sterilized scissors and sterilize them;

[0015] S1-2. Dry the sterilized small pieces on sterile filter paper, and then place them on PDA, PSA or OMA solid culture medium plates, and culture them in the dark at 28°C. After 7 days of culture, pick out single spores for purification and identification to obtain the sickle-spined spore strain.

[0016] Preferably, the disinfection method in step S1-1 is to soak the small piece in 75% ethanol solution for 1 min, then place it in 1% NaClO solution for 2 min, and then rinse it with sterile water for 3 times.

[0017] Preferably, the culture medium in step S1 is any one of PDA solid culture medium, PSA solid culture medium and OMA solid culture medium.

[0018] Preferably, the formula of the PDA solid culture medium is: 200 g potato, 20 g glucose, 18 g / L agar, and the volume is made up to 1 L with water; the formula of the PSA solid culture medium is: 200 g potato, 20 g sucrose, 18 g / L agar, and the volume is made up to 1 L with water; the formula of the OMA culture medium is: 30 g / L oatmeal, 18 g / L agar, and the volume is made up to 1 L with water.

[0019] Preferably, the activation method in step S1 is dark culturing in a solid culture medium at 28° C. for 3-4 days.

[0020] Preferably, the sterilization method of the old leaves of Yunnan pine in step S2 is high-pressure steam sterilization at 121° C. for 20 minutes, and the sterilization is repeated twice.

[0021] Preferably, the culturing method in step S3 is dark culturing at 28° C. for 7-10 days.

[0022] The present invention provides a method for rapidly and massively inducing spore production of sugarcane red rot fungus Colletotrichum fusae, which has the following advantages over the prior art:

[0023] The method of the present invention can induce conidia with good stability; can produce a large number of conidia, and provides important practical experience for the development of inducing conidia production technology of other fungi of the genus Acanthosporium; and the present invention is easy to operate, greatly improves the conidia production efficiency, shortens the inducing conidia production cycle, effectively saves manpower, material resources and a lot of precious time of scientific researchers, accelerates the research process related to sugarcane red rot, and has great application value BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a picture of a large number of conidia of Fusarium sphaerocephalum obtained by the method of Example 1 of the present invention;

[0025] Figure 2 A schematic diagram of culturing sickle-shaped spores on the solid spore-producing medium of Yunnan pine prepared in Example 1 of the present invention;

[0026] Figure 3 The bacterial colony morphology and spore production effect diagram of the strain 3 of the present invention cultured on the solid spore production medium of Yunnan pine of the present invention;

[0027] Figure 4 The colony morphology and spore production effect of the strain 3 of the present invention cultured on PDA solid medium;

[0028] Figure 5 The colony morphology and spore production effect of strain 3 of the present invention cultured on OMA solid medium;

[0029] Figure 6 The colony morphology and spore production effect of the strain 3 of the present invention cultured on PSA solid medium;

[0030] Figure 7 This is a schematic diagram of Example 1 of the present invention, in which a large number of orange-red conidia are formed by placing the mycelium blocks at the conidia-producing site on a new OMA solid culture medium after being induced by a Yunnan pine solid conidia-producing culture medium. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The Colletotrichum falciparum strains used below were isolated from diseased sugarcane midribs, sugarcane stems or sugarcane seeds of different varieties using conventional pathogen isolation methods.

[0033] The isolation method is as follows: use sterilized scissors to cut small pieces of about 3mm×5mm at the junction of diseased and healthy tissues, soak them in 75% ethanol for 1min and 1% NaClO for 2min, rinse them with sterile water for 3 times, place them on sterile filter paper to dry, and finally place them on PDA, PSA or OMA solid culture medium plates, culture them in the dark at 28℃, and after 7 days of culture, pick out single spores for purification and identification to obtain the sickle-spined spore strain.

[0034] The formula of PDA medium is: 200g potato, 20g glucose, 18g / L agar, add water to make up to 1L;

[0035] The formula of PSA medium is: 200g potato, 20g sucrose, 18g / L agar, add water to make up to 1L;

[0036] The formula of OMA culture medium is: 30g / L oatmeal, 18g / L agar, add water to make up to 1L.

[0037] Embodiment 1:

[0038] Rapid and massive induction of spore production by the sugarcane red rot pathogen Colletotrichum fusae:

[0039] 1. Place the isolated Fusarium sphaeroides strain on PDA, PSA or OMA medium and culture it in the dark at 28°C for 3 days on a clean bench for activation.

[0040] 2. Prepare a solid spore-forming medium for Yunnan pine with the following formula: KH2PO4 1.0 g / L, KNO3 1.0 g / L, MgSO4.7H2O 1.0 g / L, KCl 0.5 g / L, glucose 0.5 g / L, sucrose 0.2 g / L, and agar 18 g / L. Pour the medium into a culture dish after sterilizing with high-pressure steam at 121°C for 25 min. After the medium solidifies, put 5 to 10 old leaves of Yunnan pine that have been sterilized with high-pressure steam at 121°C for 20 min twice into each culture dish to obtain a solid spore-forming medium for Yunnan pine.

[0041] 3. Use an inoculating needle to take fresh mycelium from the edge of the colony of the above-mentioned activated strain and place it on a spore-forming medium and culture it in the dark at 28°C for 7-10 days. Under a microscope, sickle-shaped conidia can be seen in the mycelium blocks near the pine needles.

[0042] 4. Take the mycelium block where conidia are produced on the solid conidial medium of Yunnan pine and place it on a new OMA solid medium. Culture it in the dark at 28℃ for 7-10 days. A large number of orange-red conidia can be seen on the surface of the plate. The conidial yield can reach 5.5×10 5 / cm 2 -1×10 7 / cm 2 .

[0043] Comparative Example 1:

[0044] Spore production culture of sugarcane red rot pathogen Fusarium sphaeroides:

[0045] Step 1 is the same as in Example 1, and the remaining steps are:

[0046] Use an inoculation needle to take fresh mycelium from the edge of the colony of the activated strain and place it on PDA culture medium. Culture it in the dark at 28°C for 7-20 days and check the spore production under a microscope.

[0047] Comparative Example 2:

[0048] Spore production culture of sugarcane red rot pathogen Fusarium sphaeroides:

[0049] Step 1 is the same as in Example 1, and the remaining steps are:

[0050] Use an inoculation needle to take fresh mycelium from the edge of the colony of the activated strain and place it on PSA culture medium. Culture it in the dark at 28°C for 7-20 days and check the spore production under a microscope.

[0051] Comparative Example 3:

[0052] Spore production culture of sugarcane red rot pathogen Fusarium sphaeroides:

[0053] Step 1 is the same as in Example 1, and the remaining steps are:

[0054] Use an inoculation needle to take fresh mycelium from the edge of the colony of the activated strain and place it on OMA medium. Culture it in the dark at 28°C for 7-20 days and check the spore production under a microscope.

[0055] Comparative Example 4:

[0056] Spore production culture of sugarcane red rot pathogen Fusarium sphaeroides:

[0057] Step 1 is the same as in Example 1, and the remaining steps are:

[0058] Use an inoculation needle to take fresh mycelium from the edge of the colony of the activated strain and place it on PDA solid culture medium. Place 5 to 10 old leaves of Yunnan pine that have been sterilized twice with 121°C high-pressure steam for 20 minutes on the PDA solid culture medium, culture them in the dark at 28°C for 7-20 days, and check the spore production under a microscope.

[0059] Comparative Example 5:

[0060] Spore production culture of sugarcane red rot pathogen Fusarium sphaeroides:

[0061] Step 1 is the same as in Example 1, and the remaining steps are:

[0062] Use an inoculation needle to take fresh mycelium from the edge of the colony of the activated strain and place it on PDA culture medium. Incubate it in the dark at 28°C for 6 days. Remove the mycelium with a sterile cotton swab on a clean bench, rinse it twice with sterile water, dry the plate, and incubate it in the dark at 28°C for 10 days. Check the spore production under a microscope.

[0063] Detection:

[0064] 1. The activation method of Example 1 was used to activate 22 strains of Colletotrichum falciformis from different sources. The activated 22 strains were cultured according to the methods of Example 1 and Comparative Examples 1-4, respectively. The specific results are shown in Table 1 below:

[0065] Table 1

[0066]

[0067]

[0068]

[0069] Note: '++' means the conidia count is 3×10 5 / cm 2 ~5×10 5 / cm 2 ; '+' indicates that the conidia production is small, and the conidia amount is 5×10 4 / cm 2 ~2.5×10 5 / cm 2 ; “-” means no spore production.

[0070] As shown in Table 1, 22 sickle-shaped spores can stably produce conidia in 7 to 10 days using the culture method of Example 1, with a conidia production rate of 100% and a conidia amount of 3×10 5 / cm 2 ~5×10 5 / cm 2 ; Only one strain produced spores after 20 days of culture on PDA, but the spore production was small and unstable; all Fusarium spores did not produce spores on PSA; 13 strains produced 5×10 4 / cm 2 ~2.5×10 5 / cm 2 , spore production is unstable, and the amount of spores is small. It can be seen that the method of Example 1 significantly shortens the induced spore production period and the spore production is stable.

[0071] 2. The activation method of Example 1 was used to activate 10 strains of Colletotrichum falciformis, and the methods of Example 1 and Comparative Example 5 were used to induce spore production in the activated strains. The specific experimental results are shown in Table 2 below:

[0072] Table 2

[0073]

[0074] Note: '+++++' means the conidia count is 5.1×10 6 / cm 2 ~1×10 7 / cm 2 ; '++++' means the conidia amount is 1.6×10 6 / cm 2 ~5×10 6 / cm 2 ; '+++' means the conidia count is 5.5×10 5 / cm 2 ~1.5×10 6 / cm 2 ; '++' indicates that the conidia count is 3×105 / cm 2 ~5×10 5 / cm 2 ; '+' indicates that the conidia production is small, and the conidia amount is 5×10 4 / cm 2 ~2.5×10 5 / cm 2 ; “-” means no spore production.

[0075] As can be seen from Table 2 above, the method of Example 1 can obtain a large number of conidia of Spores of Spores of Spores.

[0076] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for inducing spore production of sugarcane red rot fungus Fusarium sphaerocephala, characterized in that: The inducing spore production method comprises the following steps: S1. placing the isolated Fusarium sphaeroides strain in a culture medium for activation; S2. Prepare the solid spore-forming medium of Yunnan pine: set it according to the following formula: KH2PO4 1.0g / L, KNO3 1.0g / L, MgSO4·7H2O 1.0 g / L, KCl 0.5g / L, glucose 0.5g / L, sucrose 0.2g / L, agar 18g / L, add water to make up to 1L; then sterilize it with high pressure steam at 121℃ for 25 min and pour it into the culture dish. After the culture medium solidifies, put 5-10 sterilized old leaves of Yunnan pine in each culture dish to prepare the solid spore-forming medium of Yunnan pine; S3, taking fresh mycelium from the edge of the colony of the activated strain and placing it on the solid spore-forming medium of Yunnan pine for cultivation until sickle-shaped conidia are produced in the mycelium blocks near the pine needles under a microscope; S4. Take the mycelial block where conidia are produced on the solid conidial medium of Yunnan pine and place it in OMA solid medium and culture it in the dark at 28° C. to achieve the induced conidial production of sugarcane red rot fungus Fusarium sphaerocephala.

2. A method for inducing spore production according to claim 1, characterized in that: The method for isolating and obtaining the Fusarium sphaerocephala strain comprises the following steps: S1-1. Select the diseased midrib, stem or seed of sugarcane as raw materials, cut small pieces of 3 mm × 5 mm at the junction of diseased and healthy sugarcane with sterilized scissors and sterilize them; S1-2. Dry the sterilized small pieces on sterile filter paper, and then place them on PDA, PSA or OMA solid culture medium plates, and culture them in the dark at 28°C. After 7 days of culture, pick out single spores for purification and identification to obtain the sickle-spined spore strain.

3. A method for inducing spore production according to claim 2, characterized in that: The disinfection method in step S1-1 is to soak the small piece in 75% ethanol solution for 1 minute, then place it in 1% NaClO solution for 2 minutes, and then rinse it with sterile water for 3 times.

4. A method for inducing spore production according to claim 1, characterized in that: The culture medium in step S1 is any one of PDA solid culture medium, PSA solid culture medium and OMA solid culture medium.

5. A method for inducing spore production according to claim 2, characterized in that: The formula of the PDA solid culture medium is: 200g potato, 20g glucose, 18g / L agar, and the volume is adjusted to 1L with water; the formula of the PSA solid culture medium is: 200g potato, 20g sucrose, 18g / L agar, and the volume is adjusted to 1L with water; the formula of the OMA culture medium is: 30g / L oatmeal, 18g / L agar, and the volume is adjusted to 1L with water.

6. A method for inducing spore production according to claim 1, characterized in that: The activation method in step S1 is to culture in a solid culture medium at 28° C. in the dark for 3-4 days.

7. A method for inducing spore production according to claim 1, characterized in that: The sterilization method of the old leaves of Yunnan pine in step S2 is to sterilize them with high pressure steam at 121° C. for 20 min, and repeat the sterilization twice.

8. A method for inducing spore production according to claim 1, characterized in that: The culturing method in step S3 is dark culturing at 28° C. for 7-10 days.

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