Growth promoting method of carya illinoensis scab pathogen sporophore solanacearum
By adding thin-shelled pecan fruit green peel juice to PDA culture medium and optimizing the culture conditions to pH 6 and 25℃, the problem of scab bacteria culture was solved, the growth of scab bacteria was promoted, and research progress was promoted.
Patent Information
- Application Number
- CN202510342484.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-21
- Publication Date
- 2025-07-08
AI Technical Summary
In the prior art, the cultivation of thin-shelled hickory scab bacteria is difficult to grow, which limits the research process of scab.
The PDA medium with thin-shelled pecan fruit green peel juice was used for culture. The specific conditions were pH 6 and the temperature was 25℃. 0.4% thin-shelled pecan fruit green peel juice was added to screen out the most suitable culture medium for the growth of scattered black star spores.
It significantly improved the growth rate and colony diameter of the scabbard spores, and promoted the research process of scab bacteria.
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Figure CN120272396A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of microbial growth promotion, and more specifically, relates to a method for promoting the growth of Fusicladium effusum, the pathogen of pecan scab disease. Background Art
[0002] Pecan (Carya illinoinensis (Wangenh.) K. Koch), with the commercial name "pecan", is a deciduous tree belonging to the genus Carya in the Juglandaceae family, and is native to the United States and northern Mexico. The kernels of pecan contain rich nutritional components such as vegetable oils, and it is an important woody oil tree species. China began to introduce and cultivate this tree species in the 19th century, and it has currently been promoted and planted in 14 provinces and cities such as Anhui, Yunnan, Jiangsu, and Zhejiang.
[0003] With the expansion of the pecan planting area and the increase in tree age, disease problems have become increasingly prominent, directly affecting the fruit yield. Among them, scab disease has always been the most serious yield-limiting disease in the southeastern United States. However, in the past two years, scab disease has occurred generally and severely in Jiangsu and Anhui regions of China, and about 80 - 90% of orchards are affected by scab disease. This disease can cause the fruits to be empty, shriveled, smaller in size, lower in plumpness, and poorer in quality, and in severe cases, almost result in a complete harvest failure. Therefore, scab disease has severely restricted the healthy development of the pecan industry in China.
[0004] Disease control is inseparable from the research on pathogens. After identification, the pathogen of pecan scab disease is Fusicladium effusum. It has been found that Fusicladium effusum grows slowly under various artificial culture media, which greatly limits the research progress of phytopathologists on this disease. Currently, foreign research on pecan scab disease mainly focuses on disease control and comparison of the characteristics of resistant and susceptible varieties, and there is little research on scab pathogens. The reason is related to the great difficulty in artificially culturing scab pathogens. Summary of the Invention
[0005] Aiming at the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a method for promoting the growth of Fusicladium effusum, the pathogen of pecan scab disease, for screening out an optimal culture medium that can improve the growth rate of Fusicladium effusum, the pathogen of pecan scab disease.
[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows:
[0007] A method for promoting the growth of Fusicladium effusum, the pathogen of pecan scab disease, is as follows: inoculating a Fusicladium effusum fungal cake into a culture medium supplemented with pecan fruit green peel juice for cultivation.
[0008] The culture medium is one or more of PDA medium, wheat bran medium, oat medium, Czapek medium, V8 medium, CM medium and MEA medium.
[0009] The culture medium is PDA medium.
[0010] The pH of the culture medium is 5 - 10.
[0011] The pH of the culture medium is 6.
[0012] The temperature for culturing is 10 - 35°C.
[0013] The temperature for culturing is 25°C.
[0014] The added concentration of pecan fruit green peel juice is: adding 0.2% - 2.5% of pecan fruit green peel juice to every 20 mL of the culture medium.
[0015] The added concentration of pecan fruit green peel juice is: adding 0.4% of pecan fruit green peel juice to every 20 mL of the culture medium.
[0016] A culture medium for promoting the growth of Fusicladium effusum, the formula is: PDA medium with pH = 6 + 0.4% pecan fruit green peel juice.
[0017] Compared with the prior art, the beneficial effects of the present invention are:
[0018] 1) The present invention screened the growth conditions of Fusicladium effusum in different culture media. The results showed that the hyphae of Fusicladium effusum could grow on PDA, wheat bran, OMA, Czapek, V8, CM and MEA media, but grew fastest on PDA medium.
[0019] 2) The present invention screened the growth conditions of Fusicladium effusum at different pH values. The results showed that the hyphae of Fusicladium effusum could grow on PDA medium with pH 5 - 10, and grew fastest on PDA medium with pH = 6.
[0020] 3) The present invention screened the growth conditions of Fusicladium effusum at different temperatures. The results showed that the hyphae of Fusicladium effusum could grow at 10 - 35°C, and grew fastest at 25°C.
[0021] 4) The present invention screened the growth conditions of Fusicladium effusum in different carbon sources. The results showed that the hyphae of Fusicladium effusum could grow in the culture medium added with glucose, mannitol, glycerol, sorbitol and β - lactose, but the most suitable carbon source for the growth of Fusicladium effusum hyphae is glucose.
[0022] 5) The growth conditions of Fusicladium effusum in different nitrogen sources were screened in the present invention. The results showed that the hyphae of Fusicladium effusum could grow in the culture media supplemented with sodium nitrate, peptone, and potassium nitrate, and the most suitable nitrogen source for the growth of Fusicladium effusum hyphae was sodium nitrate.
[0023] 6) The growth conditions of Fusicladium effusum in different addition amounts of Carya illinoinensis pericarp juice were screened in the present invention. The results showed that the hyphae of Fusicladium effusum grew well on the PDA medium (pH = 6) supplemented with 0.2 - 2.5% of Carya illinoinensis pericarp juice per 20 mL. Among them, the growth was the fastest on the PDA medium supplemented with 0.4% of Carya illinoinensis pericarp juice, and the average diameter of the colony was 4.95 cm. Description of the Drawings
[0024] Figure 1 Growth conditions of Fusicladium effusum on different culture media (A is PDA medium; B is wheat bran medium; C is OMA medium; D is Czapek medium; E is V8 medium; F is CM medium; G is MEA medium);
[0025] Figure 2 Growth conditions of Fusicladium effusum growing on PDA media with different pH values (A is pH = 5; B is pH = 6; C is pH = 7; D is pH = 8; E is pH = 9; F is pH = 10);
[0026] Figure 3 Growth conditions of Fusicladium effusum at different temperatures (A is 5°C; B is 10°C; C is 15°C; D is 20°C; E is 25°C; F is 30°C; G is 35°C);
[0027] Figure 4 Growth conditions of Fusicladium effusum in different carbon sources (A is glucose; B is mannitol; C is glycerol; D is sorbitol; E is β - lactose; F is arabinose; G is D - galactose; H is xylitol);
[0028] Figure 5 Growth conditions of Fusicladium effusum in different nitrogen sources (A is sodium nitrate; B is peptone; C is potassium nitrate; D is sodium nitrite; E is urea);
[0029] Figure 6 Growth conditions of Fusicladium effusum on the PDA medium with pH = 6 supplemented with different volumes of Carya illinoinensis pericarp juice (A is 0.2%; B is 0.4%; C is 0.5%; D is 0.75%; E is 1%; F is 2.5%; G is 5%). Detailed Embodiments
[0030] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are all conventional means well known to those skilled in the art.
[0031] The pathogenic bacterium used in this application is the commercially available Fusicladium effusum, which is the pathogenic bacterium of pecan scab and has an important impact on the yield and quality of pecan. It was purchased from Ningbo Taisituo Biotechnology Co., Ltd.
[0032] The pecan fruit green peel material used in this application was collected from the immature fruit green peel of the 'Pawnee' variety in the pecan experimental base of the research group where the applicant is located.
[0033] Example 1
[0034] 1. Growth of Fusicladium effusum on different culture media
[0035] 1) Potato dextrose agar (PDA) medium
[0036] 200 g of fresh peeled potatoes were cut into small pieces and put into a pot, added 1 L of water, heated to boiling in a heater. After the potatoes were completely cooked, filtered through 2 - 3 layers of gauze while it was hot on a measuring cup, discarded the filter residue, then added 20 g of glucose, 15 - 20 g of agar, and made up the filtrate with water to 1 L. After being dispensed into Erlenmeyer flasks, it was sterilized at 121 °C under high-pressure steam for 20 min.
[0037] 2) Wheat bran medium
[0038] 200 g of fresh peeled potatoes were cut into small pieces and put into a pot, added 1 L of water, heated to boiling in a heater. After the potatoes were completely cooked, filtered through 2 - 3 layers of gauze while it was hot on a measuring cup, discarded the filter residue, then added 20 g of glucose, 10 g of wheat bran powder, 10 g of soybean powder, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 15 - 20 g of agar, adjusted the pH to 7, and made up the water to 1 L. After being dispensed into Erlenmeyer flasks, it was sterilized at 121 °C under high-pressure steam for 20 min. Each treatment was repeated 5 times.
[0039] 3) Oatmeal (OMA) medium
[0040] 40 g of oats, 5 g of sucrose, 15 - 20 g of agar, and deionized water were used to prepare 1 L of medium, sterilized at 121 °C under high-pressure steam for 20 min, and each treatment was repeated 5 times.
[0041] 4) Czapek Dox Medium
[0042] 2 g of sodium nitrate, 1 g of dipotassium hydrogen phosphate, 0.5 g of potassium chloride, 0.5 g of magnesium sulfate, 0.01 g of ferrous sulfate, 30 g of sucrose, 15 - 20 g of agar, and deionized water are used to prepare 1 L of culture medium, which is autoclaved at 121 °C for 20 min, and each treatment is repeated 5 times.
[0043] 5) V8 medium
[0044] Take 500 mL of distilled water, add 20 g of agar, and autoclave at 121 °C for 15 min; take 500 mL of V8 juice medium (2× solution), and incubate in a water bath at 50 - 60 °C for 30 min; mix the above two solutions evenly and pour them into petri dishes in time. Each treatment is repeated 5 times.
[0045] 6) CM medium
[0046] 20 g of sucrose, 6 g of yeast extract, 6 g of Casamino acid, 15 - 20 g of agar, add water to make up to 1 L. After dispensing into Erlenmeyer flasks, autoclave at 121 °C for 20 min. Each treatment is repeated 5 times.
[0047] 7) Malt extract (MEA) medium
[0048] 30 g of malt extract, 3 g of soy peptone, 15 g - 20 g of agar, add distilled water to prepare 1 L. After making into a solution, sterilize at 121 °C for 20 min, and adjust the pH to 5.6 ± 0.2. Each treatment is repeated 5 times.
[0049] Place the 5 - mm - diameter Pyricularia aciculata discs in the center of the above - mentioned culture medium plates respectively, culture them in an environment at 25 °C for 40 days, then observe the mycelial growth situation, measure and count the colony diameters of Pyricularia aciculata on different culture media, and determine the colony diameters by the cross - method.
[0050] The results are shown in Table 1. The mycelia of Pyricularia aciculata can grow on PDA, wheat bran, OMA, Czapek, V8, CM, and MEA media, but grow fastest on the PDA medium ( Figure 1 ).
[0051] Table 1 Growth of Pyricularia aciculata on different culture media
[0052]
[0053] 2. Growth status of Pyricularia aciculata on PDA media with different pH values
[0054] Cut 200 g of fresh peeled potatoes into small pieces and put them into a pot. Add 1 L of water and heat to boiling. After the potatoes are completely cooked, filter them through 2 - 3 layers of gauze while it is still hot into a measuring cup. Discard the residue, then add 20 g of glucose and make up the filtrate with water to 1 L. Dispense it into Erlenmeyer flasks at 100 mL per flask, and then adjust the pH to 5, 6, 7, 8, 9, 10 respectively with 1 mol / L HCl and NaOH. Add 1.5 - 2 g of agar to each medium and sterilize at 121 °C under high pressure steam for 20 min to prepare the PDA medium. Repeat each treatment 5 times. Place a 5 - mm diameter Pyricularia aceris Sacc. disc in the center of the PDA medium plates with different pH values, and observe the mycelial growth after culturing for 40 days at 25 °C. Measure the colony diameter using the cross - method.
[0055] The results are shown in Table 2. The mycelia of Pyricularia aceris Sacc. can grow on PDA media with pH values ranging from 5 to 10, and among them, the growth is the fastest on the PDA medium with pH = 6 ( Figure 2 ).
[0056] Table 2 Growth of Pyricularia aceris Sacc. on PDA media with different pH values
[0057]
[0058] 3. Growth status of Pyricularia aceris Sacc. at different temperatures
[0059] Place a 5 - mm diameter Pyricularia aceris Sacc. disc in the center of the PDA medium plate with pH = 6, and incubate it at a constant temperature of 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C and 35 °C for 30 d, then observe the mycelial growth. Repeat each treatment 5 times. Measure and count the colony diameters of Pyricularia aceris Sacc. cultured at different temperatures, and measure the colony diameter using the cross - method.
[0060] The results are shown in Table 3. The growth of Pyricularia aceris Sacc. mycelia is severely inhibited at 5 °C, and it can grow at other temperatures. Among them, the growth is the fastest at 25 °C ( Figure 3 ).
[0061] Table 3 Growth of Pyricularia aceris Sacc. at different culture temperatures
[0062]
[0063] 4. Growth of Pyricularia aceris Sacc. in different carbon sources
[0064] Based on Czapek medium, replace the carbon source (sucrose) with glucose, mannitol, glycerol, sorbitol, β - lactose, arabinose, D - galactose, xylitol in equal amounts respectively to make media with different carbon sources. Repeat each treatment 5 times.
[0065] Place the scattered Phyllosticta circular discs with a diameter of 5 mm at the center of the above-mentioned culture medium plates respectively, and observe the mycelial growth after culturing for 40 days in an environment of 25 °C. Measure and count the colony diameters of the scattered Phyllosticta on the culture media with different carbon sources, and determine the colony diameters by the cross method.
[0066] The results are shown in Table 4. The mycelia of the scattered Phyllosticta can grow in the culture media supplemented with glucose, mannitol, glycerol, sorbitol and β-lactose, but the most suitable carbon source for the growth of the scattered Phyllosticta mycelia is glucose; the growth is severely inhibited in the culture media supplemented with arabinose, D-galactose and xylitol ( Figure 4 ).
[0067] Table 4 Growth of the scattered Phyllosticta in different carbon sources
[0068]
[0069] 5. Growth of the scattered Phyllosticta in different nitrogen sources
[0070] Based on the Czapek medium, replace the nitrogen source (sodium nitrate) in it with sodium nitrite, urea, peptone, potassium nitrate in equal amounts respectively to prepare the culture media with different nitrogen sources, and repeat each treatment 5 times.
[0071] Place the scattered Phyllosticta circular discs with a diameter of 5 mm at the center of the above-mentioned culture medium plates respectively, and observe the mycelial growth after culturing for 40 days in an environment of 25 °C. Measure and count the colony diameters of the scattered Phyllosticta on the culture media with different nitrogen sources, and determine the colony diameters by the cross method.
[0072] The results are shown in Table 5. The mycelia of the scattered Phyllosticta can grow in the culture media supplemented with sodium nitrate, peptone and potassium nitrate. The most suitable nitrogen source for the growth of the scattered Phyllosticta mycelia is sodium nitrate, but the growth is severely inhibited in the culture media supplemented with sodium nitrite and urea respectively ( Figure 5 ).
[0073] Table 5 Growth of the scattered Phyllosticta in different nitrogen sources
[0074]
[0075] 6. Effect of the pericarp juice of Carya illinoinensis fruits on the growth of the scattered Phyllosticta
[0076] Weigh 20 g of the pericarp of Carya illinoinensis fruits. After grinding it in liquid nitrogen, add ethanol for suspension. After ultrasonic treatment in an ultrasonic device for 5 min, centrifuge at 12,000 rpm for 5 min. Take the supernatant, filter it through a 0.22-µM bacterial filter, and collect it. Add 0.2%, 0.4%, 0.5%, 0.75%, 1%, 2.5%, and 5% of the pericarp juice of Carya illinoinensis to 20 mL of PDA medium (pH = 6) respectively, that is, the addition amounts are (40 µL, 80 µL, 100 µL, 150 µL, 200 µL, 500 µL, and 1 mL) respectively, and repeat each treatment 5 times.
[0077] Place the scattered Venturia nashicola fungus cakes with a diameter of 5 mm at the center of the above-mentioned culture medium plates respectively, and observe the mycelial growth after culturing in an environment of 25 °C for 40 days. Measure and count the colony diameters of the scattered Venturia nashicola on the culture media with different addition amounts of the pericarp juice of Carya illinoinensis, and measure the colony diameters by the cross method.
[0078] The results are shown in Table 6. The mycelia of the scattered Venturia nashicola grow well on the PDA medium (pH = 6) with 0.2%, 0.4%, 0.5%, 0.75%, 1%, and 2.5% of the pericarp juice of Carya illinoinensis added to 20 mL respectively, but the growth is severely inhibited on the PDA medium with 5% of the pericarp juice of Carya illinoinensis added. Among them, the growth is the fastest on the PDA medium with 0.4% of the pericarp juice of Carya illinoinensis added, and the average colony diameter is 4.95 cm ( Figure 6 )
[0079] Table 6 Growth of the scattered Venturia nashicola on the PDA medium with different addition amounts of the pericarp juice of Carya illinoinensis
[0080]
[0081] The above description is only illustrative and not restrictive for the present invention. Those of ordinary skill in the art understand that many modifications, variations, or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all of them will fall within the protection scope of the present invention.
Claims
1. A method for promoting the growth of *Fusicladium effusum* causing pecan scab, characterized in that, The scattered Fusicladium effusum cake is inoculated into a medium supplemented with Carya illinoinensis fruit green peel juice for cultivation.
2. The growth-promoting method according to claim 1, wherein The medium is one or several of PDA medium, wheat bran medium, oat medium, Czapek medium, V8 medium, CM medium and MEA medium.
3. The growth-promoting method according to claim 2, characterized in that, The medium is PDA medium.
4. The growth-promoting method according to claim 1, wherein The pH of the medium is 5 - 10.
5. The growth-promoting method according to claim 4, wherein The pH of the medium is 6.
6. The growth-promoting method according to claim 1, characterized in that, The temperature of the cultivation is 10 - 35 °C.
7. The growth-promoting method according to claim 6, wherein The temperature of the cultivation is 25 °C.
8. The growth promotion method according to claim 1, characterized in that, The added concentration of the Carya illinoinensis fruit green peel juice is: 0.2% - 2.5% of the Carya illinoinensis fruit green peel juice is added to every 20 mL of the medium.
9. The growth-promoting method according to claim 8, characterized in that, The added concentration of the Carya illinoinensis fruit green peel juice is: 0.4% of the Carya illinoinensis fruit green peel juice is added to every 20 mL of the medium.
10. A culture medium for promoting the growth of Fusicladium effusum, characterized in that, The formula is: PDA medium with pH = 6 + 0.4% Carya illinoinensis fruit green peel juice.