CN109402009B

CN109402009B - Method for screening nitrogen-fixing blue algae antagonistic to rhizoctonia solani and application thereof

Info

Publication number: CN109402009B
Application number: CN201811365944.3A
Authority: CN
Inventors: 贺鸿志; 周伊薇; 包江桥; 卢玉真; 卓晨; 黎华寿
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2018-11-16
Filing date: 2018-11-16
Publication date: 2021-03-16
Anticipated expiration: 2038-11-16
Also published as: CN109402009A

Abstract

The invention discloses a method for screening nitrogen-fixing blue algae antagonistic to rhizoctonia solani and application thereof. The invention can realize better growth of the rhizoctonia solani and the nitrogen-fixing blue algae in the same culture dish by optimizing the culture medium, realizes the interaction of the rhizoctonia solani and the nitrogen-fixing blue algae under the simulated actual condition, and can directly observe the growth confrontation condition of the algae and the rhizoctonia solani. The method provided by the invention can screen 2 algae in 4-grid culture dishes at the same time, and can screen 4-8 algae in more grid culture dishes at the same time. Compared with 1 plate of the traditional extracting solution plate growth inhibition experiment, only 1 algae can be screened and the effective components of the algae must be extracted for the experiment. Therefore, the invention greatly improves the screening efficiency and reduces the environmental pollution. Meanwhile, more importantly, the experimental result is closer to the interaction condition of the phycomycetes under the actual condition, so that the screening effectiveness is stronger.

Description

Method for screening nitrogen-fixing blue algae antagonistic to rhizoctonia solani and application thereof

Technical Field

The invention relates to the field of biological prevention and control of plant diseases and the field of microorganism screening, in particular to a method for screening nitrogen-fixing blue algae antagonistic to rhizoctonia solani and application thereof.

Background

The rice sheath blight disease is a soil-borne fungal disease caused by fungal infection. The pathogen is the panzephyrus cucumeris (Frank) donk, which belongs to the basidiomycotina, and the asexual Rhizoctonia solani Kuhn belongs to the deuteromycotina. Sclerotia formed by pathogenic bacteria of rice sheath blight disease have strong stress resistance, can survive in soil for a long time, can be transversely spread by means of plant leaves during disease attack, and has soil-borne and leaf-borne capabilities, so that the sclerotia becomes refractory diseases (Yangyingqing, etc., 2014) which are difficult to control. In recent years, with the application and popularization of high-yield, short-stalk, multi-tiller and fertilizer-resistant rice varieties and the change of farming modes and climatic conditions, rice sheath blight diseases are increasingly serious, and the rice sheath blight diseases are one of the main diseases in the current rice production (xiezonghua and the like, 2012). For preventing and treating the rice sheath blight, at present, the method mainly depends on planting disease-resistant varieties and applying chemical pesticides. Because of wide pathogenic bacteria host range, no rice germplasm immune to banded sclerotial blight has been found so far, resistant varieties are rare, and difficulty is increased for disease-resistant breeding (Srinivasachary et al, 2011). The resistance of disease-resistant varieties is easy to degrade, and the general resistance of high-quality and high-yield varieties is lower. Validamycin is a specific drug for banded sclerotial blight in China, is a main prevention and treatment drug for rice fields, but has high potential ecological environment and human health risk when being singly used for preventing and treating validamycin for a long time (Mew et al, 2004). The european union has formally banned the sale of validamycin since 2004. The method searches for biocontrol bacteria resources, utilizes beneficial organisms at the rhizosphere of plants, explores an environment-friendly and low-cost biological control method for preventing and treating rice sheath blight, and has important significance for agricultural sustainable development (Khokhar et al, 2012). In recent years, researchers at home and abroad have found some fungi, bacteria and actinomycetes having antagonistic action against rhizoctonia solani, a pathogenic bacterium of rice sheath blight disease (Nagarajan et al, 2013). However, the variability of the biocontrol bacteria and the influence of various complex environments and biological factors on the field application thereof cause unstable control effect and prevent large-scale commercial application thereof, so that further research and development of the biocontrol bacteria with better effect are urgently needed.

After 1939 Indian scientists report that blue-green algae is used for fertilizing the field for the first time, related researches are carried out in many countries successively, and the researches prove that the yield can be increased by inoculating the blue-green algae in a rice field. The rice field algae cultivation test is carried out in partial provinces of south China at the beginning of Li Shanghao in the last 50 th century, and the result shows that the rice yield is increased by 10-30% (Li Shanghao et al, 1962). Today, global warming and water eutrophication are becoming more serious, and the development of low-carbon agriculture of paddy fields and the control of agricultural non-point source pollution by utilizing biodiversity have important significance (Jackson et al, 2007). The biological nitrogen fixation by using the blue algae is just suitable for the trend. Blue algae bred in the rice field can not only provide nitrogen (nitrogen fixation) and organic matters (carbon fixation) for crops, improve the yield, reduce the fertilizer dosage and the production cost, improve the phosphate solubility and reduce water and soil pollution, but also supply oxygen to the roots of the crops, secrete growth regulators such as plant hormones, amino acids, vitamins and the like and inhibit the growth of weeds, thereby achieving the purposes of maintaining ecological balance and promoting agricultural sustainable development (Prasanna et al, 2013; Singh et al, 2011). While Singh et al (2014) and others believe that blue-green algae may also play an important role in crop protection, there have been some reports on blue-green algae inhibiting rhizoctonia solani pathogenic bacteria, and laboratory studies of Chaudhary et al (2012) and others show that certain kinds of culture solutions or algae cell extracts of nostoc and anabaena can inhibit many harmful microorganisms including r.

The classical method for evaluating microbial competition is generally plate confrontation experiment, and the interaction between two or more kinds of microbes can be clearly observed through the confrontation experiment. However, the culture medium formulations of algae and bacteria are very different, and therefore, the growth counterexperiment cannot be performed on the same plate. The existing method for screening algae which antagonizes pathogenic microorganisms generally adopts a plate growth inhibition method of an algae extract or an extracellular secretion, and the method can only indirectly judge the interaction effect of the algae and the microorganisms. In addition, the experiment needs to culture algae in advance, prepare algae extracts or extracellular secretions, add the effective components of algae extracted by different solvents or extracellular secretions into a culture medium suitable for the growth of pathogenic microorganisms to prepare a flat plate, the operation process is complex, the screening flux is low, and pollution is caused by the extraction by using an organic solvent.

Disclosure of Invention

The invention aims to overcome the defect that the prior art can not evaluate the direct interaction of algae and bacteria, and provides a method for screening nitrogen-fixing blue-green algae for antagonizing rhizoctonia solani.

The invention also aims to provide the application of the method for screening the nitrogen-fixing blue algae for antagonizing rhizoctonia solani.

The purpose of the invention is realized by the following technical scheme: a method for screening nitrogen-fixing blue algae for antagonizing rhizoctonia solani comprises the following steps:

(1) BG11 were prepared separately₀Solid culture medium and PDA solid culture medium; the sterilized PDA solid culture medium which is still in liquid state and BG11₀Mixing the solid culture mediums to obtain a mixed culture medium;

(2) respectively pouring a mixed culture medium and a PDA solid culture medium into the lattices of the sterile culture dish with the lattices, wherein the mixed culture medium and the PDA solid culture medium are adjacent; the height of the mixed culture medium and the PDA solid culture medium in the culture dish is the same as that of partitions forming grids in the culture dish;

(3) inoculating algae to be screened in a central area of the sub-standard cells poured with the mixed culture medium, culturing in an artificial climate box until the algae to be screened stably grows on the culture medium, and then inoculating rhizoctonia solani in the central area of the sub-standard cells poured with the PDA solid culture medium;

(4) culturing; observing and measuring when the rhizoctonia solani in the control culture dish grows over the culture dish, if the algae can normally grow and can inhibit the diffusion of pathogenic bacteria to the area where the algae are located, judging that the algae has the capacity of antagonizing the rhizoctonia solani, judging the bacteriostasis strength according to the size of a bacteriostasis zone, and if the rhizoctonia solani is covered by the rhizoctonia solani, indicating that the resistance does not exist, as shown in figure 1.

BG11 described in step (1)₀The composition of the solid medium was as follows: k₂HPO₄.3H₂O 0.04g/L、MgSO₄.7H₂O 0.075g/L、CaCl₂.2H₂O0.036 g/L, citric acid 0.006g/L, ferric ammonium citrate 0.006g/L, EDTA 0.001.001 g/L, Na₂CO₃0.02g/L, trace element A51 mL/L and agar 15-20 g/L;

the composition of trace element a5 is as follows: h₃BO₃ 2.860g/L、NaMoO₄.2H₂O 0.021g/L、MnCl₂.4H₂O 1.810g/L、ZnSO₄.7H₂O 0.222g/L、CuSO₄.5H₂O 0.079g/L、NiSO₄.6H₂O 0.479g/L。

The PDA solid culture medium in the step (1) comprises the following components: 200g of fresh potato peeled, chopped and boiled extract, 20g of glucose, 15-20 g of agar, and pure water with constant volume of 1L and natural pH.

The sterilization condition in the step (1) is preferably sterilization at 115-121 ℃ for 15-30 min; more preferably, the sterilization is carried out for 15-20 min at 121 ℃.

PDA culture medium described in step (1) andBG11₀The culture medium is prepared according to the following steps of 1: 2-18 parts by weight; more preferably, the mixing is carried out in a volume ratio of 1: 10.

The culture dish in the step (2) is preferably a culture dish with a diameter of 90 mm.

The inoculation amount of the algae in the step (3) is preferably calculated according to the amount of formed algae clusters with the diameter of 3-10 mm; more preferably, it is calculated as the amount of algal mass formed with a diameter of 5 mm.

The preferable conditions of the culture in the step (3) are 23-30 ℃ and illumination of 2500-3200 lx for 3-5 days; more preferably, the culture is carried out at 25-28 ℃ under illumination of 3000lx for 4 days.

The inoculation amount of the rhizoctonia solani in the step (3) is preferably calculated according to the rhizoctonia solani with the inoculation diameter of 3-8 mm; more preferably, it is calculated by inoculating Rhizoctonia solani of 5mm in diameter.

The culture condition in the step (4) is preferably culture at 23-30 ℃ and illumination of 2500-3200 lx; more preferably, the culture is carried out at 25 to 28 ℃ and 3000 lx.

The method for screening the nitrogen-fixing blue algae antagonistic to rhizoctonia solani is applied to screening the nitrogen-fixing blue algae antagonistic to rhizoctonia solani.

Compared with the prior art, the invention has the following advantages and effects:

the invention can realize the optimal growth of two types of microorganisms with greatly different culture conditions in the same culture dish by optimizing the culture medium, realizes the interaction of the two types of microorganisms under the simulated actual condition, and can directly observe the growth opposite conditions of algae and rhizoctonia solani. Meanwhile, 2 algae can be screened in 4-grid culture dishes simultaneously by using the method, and 4-8 algae can be screened simultaneously by using more grid culture dishes. Compared with 1 plate of the traditional extracting solution plate growth inhibition experiment, only 1 algae can be screened and the effective components of the algae must be extracted for the experiment. Therefore, the method provided by the invention greatly improves the screening efficiency and reduces the environmental pollution. Meanwhile, more importantly, the experimental result is closer to the interaction condition of the phycomycetes under the actual condition, so that the screening effectiveness is stronger.

Drawings

FIG. 1 is a diagram of the present inventionManaging the graph; wherein, the graph A is a culture dish with four divisions, and the graph B is a culture dish with 5 divisions; a. the₁～A₄Represents different blue algae, R represents rhizoctonia solani, P represents PDA culture medium, B represents BG11₀And (4) a culture medium.

Fig. 2 is a diagram of different PDAs: BG11₀Photograph of growth of Rhizoctonia solani on the culture medium in volume ratio.

Fig. 3 is a diagram of different PDAs: BG11₀The growth status of two representative nitrogen-fixing blue algae on the proportioned culture medium is shown in the picture.

FIG. 4 is a photograph showing the results of the confronting experiment of nitrogen-fixing cyanobacteria and Rhizoctonia solani on the mixed culture medium; where the 1 st plate is the PDA control.

FIG. 5 is a photograph of an experimental cement pool.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Materials used in the examples:

blue-green algae: nostoc sp (Nostoc sp.) FACHB-85, Nostoc paludosum (Nostoc paludosum) FACHB-89, Nostoc sp (Nostoc sp.) FACHB-131, Anabaena variabilis (Anabaena variabilis) FACHB-164 and Anabaena paddybaena (Anabaena oryzae) FACHB-604, of the freshwater algae seed bank of the Chinese academy of sciences; the laboratory separates and purifies the preserved strain Nostoc piscinale SCAU-003 (CCTCC NO: M2018194, the preservation date is 2018, 4 and 11 days, the preservation unit is the China center for type culture Collection located in the university of Wuhan, China, and is disclosed in patent application 201810397659.3).

Rhizoctonia solani, number GIM3.512, purchased from Guangdong institute for microbiology, and used as a pathogenic bacterium of rice sheath blight.

Example 1: screening of Mixed Medium

(1) BG11 containing 1.5% agar was prepared separately₀Solid culture medium and PDA solid culture medium; the PDA solid culture medium and BG11 which are in liquid state after sterilization (sterilization at 121 ℃ for 15min) are added₀Mixing the solid culture medium at different volume ratios (1:2, 1:6, 1:10,1:14, 1:18) to obtain a mixed culture medium.

(2) The PDA solid culture medium obtained in the step (1) and BG11₀The solid culture medium and the mixed culture medium with different proportions are poured into a flat plate, microalgae and rhizoctonia solani are respectively inoculated on the flat plate, and the culture is carried out at 28 ℃ and under 3000lx illumination environment.

(3) The experimental result shows that the 6 blue algae can not grow normally on pure PDA culture medium, while the rhizoctonia solani GIM3.512 can grow normally on BG11₀The growth effect on the medium was also poor. PDA Medium and BG11₀Compared with a control, the mixed culture medium obtained by the culture medium according to the proportion in the step (1) can cover most of the area of a culture dish except 1:14 and 1:18, as shown in figure 2, a PDA culture medium and the mixed culture medium in figure 2 are sequentially arranged in a culture dish with 4 lattices and a diameter of 9cm, the mixed culture medium is arranged between the PDA culture medium and the PDA culture medium, rhizoctonia solani is inoculated on the PDA, and the PDA culture medium is arranged in 4 lattices of a control group (CK). The results in FIG. 2 illustrate that PDA and BG11 were shown to favor the growth of Rhizoctonia solani₀The ratio of the culture medium is 1: 0-10. Meanwhile, considering the adverse effect of PDA medium on the growth of algae, the growth conditions of two typical nitrogen-fixing cyanobacteria (Nostoc sp. FACHB-131 and Anabaena variabilis FACHB-164) on mixed media of different proportions were evaluated, and the results are shown in FIG. 3, which indicate that two algae grow significantly more than pure BG11 on two mixed media of 1:2 and 1:6₀Control Difference in Medium, algal growth on 1:10 Mixed Medium and BG11₀The comparison shows that for nitrogen-fixing blue algae, PDA and BG11₀The ratio of the culture medium is 1: 10-18. Therefore, PDA and BG11 were all selected subsequently₀The proportion of the culture medium is 1: 10.

Example 2: screening of microalgae antagonistic to Rhizoctonia solani

(1) The preparation of the medium was the same as in step (1) of example 1, except that the mixed medium was PDA medium and BG11₀The liquid culture media are mixed in a volume ratio of 1: 10.

(2) And pouring the culture medium into the four-grid culture dish in the grid, pouring the mixed culture medium into the 2 grids, and pouring the PDA culture medium into the 2 grids, wherein the mixed culture medium and the mixed culture medium are separated by the PDA culture medium. The height of the culture medium in the cells is consistent with the height of the partitions between the cells.

(3) Will be in BG11 in advance₀Inoculating algae cultured in solid culture medium into the cell center region containing mixed culture medium in the plate of step (2), culturing at 28 deg.C and illumination intensity of 3000lx for 4 days with diameter of algae mass of 5 mm. Then inoculating activated rhizoctonia solani (fungus cake with diameter of 5 mm) in the cell center area containing PDA culture medium in the plate of the step (2). The cultivation was continued under the same illumination and temperature.

(4) And observing whether the zone where the algae is close to the bacteria has an antibacterial zone after the hyphae in the control plate grows over the plate. If the zone of inhibition is present, the width of the zone is measured, and the antagonistic capacity of the algae is determined by the width of the zone.

The final experimental results are shown in FIG. 4, the 1 st dish on the left is a Control (CK), the other 3 dishes are opposite experimental plates of algae and rhizoctonia solani, and one algae strain is respectively arranged on the left lower part and the right upper part of each dish; the results show that the algae SCAU-003 has obvious inhibition zones around, while the FACHB-85, FACHB-131 and FACHB-89 have no inhibition zones but have no hypha coverage completely, while the FACHB-604 and FACHB-164 are completely covered by rhizoctonia solani.

Example 3: cement pond verification experiment for antagonizing nitrogen-fixing blue algae of rhizoctonia solani

(1) And 3-8 months in 2017, wherein the place is an ecological farm of southern China agricultural university. According to the above plate experiment results, 4 different effect algae (strain SCAU-003, FACHB-85, FACHB-89 and FACHB-604) treatments and 1 control CK without algae were selected, each group was repeated three times, and 15 cement ponds (1m × 1m) were constructed.

(1) BG11 in laboratory₀4 kinds of algae are cultured in a culture medium, the spun silk is filtered and collected, the mixture is weighed and diluted to 500ml with water and then is scattered into a pool when the tillering of rice (Huanghuazhan, purchased from Shenzhen Longjinguo Setaria, Ltd.) is started. 6g of wet algae were added to each cement pond. After the algae grows for 1 week, 50mL of rhizoctonia solani inoculum which is aseptically cultured for 12 days by using a PDB liquid medium is added. The PDB culture medium formula is as follows: 200The fresh potato is peeled, cut and boiled extract, glucose is 20g, pure water is 1L, agar is not added, and the pH value is natural. And (5) investigating disease grade 30 days after the rice ears. See figure 5 for the experimental site.

Experimental results show that the effect of inhibiting rice sheath blight by stocking SCAU-003 in a cement pond environment is obvious, compared with CK inhibition rate of 37%, both FACHB-85 and FACHB-89 have certain inhibition effects, and FACHB-604 shows that the development of the rice sheath blight is promoted, which is consistent with the results of the flat plate experiment. This shows that the screening result of the plate confrontation method provided by the invention is reliable.

TABLE 1 Cement pond nitrogen fixation blue algae disease resistance experiment result

Note: CK is a reference, and the negative number indicates the invasion of the microorganism promoting bacteria compared with CK

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A method for screening nitrogen-fixing blue algae for antagonizing rhizoctonia solani is characterized by comprising the following steps:

(2) respectively pouring a mixed culture medium and a PDA solid culture medium into the lattices of a sterile culture dish with lattices, and respectively pouring a culture medium into the adjacent lattices; the mixed culture medium is adjacent to the PDA solid culture medium; the height of the mixed culture medium and the PDA solid culture medium in the culture dish is the same as that of partitions forming grids in the culture dish;

(4) culturing; observing and measuring when the rhizoctonia solani in the contrast culture dish grows over the culture dish, if the algae can normally grow and can inhibit the diffusion of pathogenic bacteria to the area where the algae are located, judging that the algae has the capacity of antagonizing the rhizoctonia solani, judging the bacteriostasis strength according to the size of a bacteriostasis zone, and if the rhizoctonia solani is covered by the rhizoctonia solani, indicating that the resistance does not exist;

BG11 described in step (1)₀The composition of the solid medium was as follows: k₂HPO₄.3H₂O 0.04 g/L、MgSO₄.7H₂O 0.075 g/L、CaCl₂.2H₂O0.036 g/L, citric acid 0.006g/L, ferric ammonium citrate 0.006g/L, EDTA 0.001.001 g/L, Na₂CO₃0.02g/L, trace element A51 mL/L and agar 15-20 g/L;

the composition of trace element a5 is as follows: h₃BO₃2.860 g/L、NaMoO₄.2H₂O 0.021 g/L、MnCl₂.4H₂O 1.810 g/L、ZnSO₄.7H₂O 0.222 g/L、CuSO₄.5H₂O 0.079 g/L、NiSO₄.6H₂O0.479 g/L; the PDA culture medium and the BG11 in the step (1)₀The culture medium is prepared according to the following steps of 1: 2-18 parts by weight.

2. The method for screening nitrogen-fixing cyanobacteria for antagonizing rhizoctonia solani according to claim 1, wherein the method comprises the following steps: the PDA culture medium and BG11₀The culture mediums are mixed according to the volume ratio of 1: 10.

3. The method for screening nitrogen-fixing cyanobacteria for antagonizing rhizoctonia solani according to claim 1, wherein the method comprises the following steps:

the sterilization condition in the step (1) is sterilization at 115-121 ℃ for 15-30 min;

the culture dish in the step (2) is a culture dish with the diameter of 90 mm.

4. The method for screening nitrogen-fixing cyanobacteria for antagonizing rhizoctonia solani according to claim 1, wherein the method comprises the following steps:

calculating the inoculation amount of the algae in the step (3) according to the amount of formed algae clusters with the diameter of 3-10 mm;

and (4) calculating the inoculation amount of the rhizoctonia solani in the step (3) according to the rhizoctonia solani with the inoculation diameter of 3-8 mm.

5. The method for screening nitrogen-fixing cyanobacteria for antagonizing rhizoctonia solani according to claim 4, wherein the method comprises the following steps:

calculating the inoculation amount of the algae in the step (3) according to the amount of formed algae clusters with the diameter of 5 mm;

the inoculation amount of the rhizoctonia solani in the step (3) is calculated according to the rhizoctonia solani with the inoculation diameter of 5 mm.

6. The method for screening nitrogen-fixing cyanobacteria for antagonizing rhizoctonia solani according to claim 1, wherein the method comprises the following steps:

the culture condition in the step (3) is that the culture is carried out for 3-5 days at 23-30 ℃ and 2500-3200 lx;

the culture condition in the step (4) is culture at 23-30 ℃ and 2500-3200 lx.

7. The method for screening nitrogen-fixing cyanobacteria for antagonizing rhizoctonia solani according to claim 6, wherein the method comprises the following steps:

culturing for 4 days at the temperature of 25-28 ℃ and 3000lx under the culturing condition in the step (3);

the culture condition in the step (4) is 3000lx culture at 25-28 ℃.

8. The method for screening nitrogen-fixing blue-green algae antagonistic to rhizoctonia solani according to any one of claims 1 to 7, and the application of the method in screening nitrogen-fixing blue-green algae antagonistic to rhizoctonia solani.