CN114907992B - Strain C11 antagonizing plant pathogenic bacteria and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biocontrol microorganisms, and particularly relates to a strain C11 for antagonizing plant pathogenic bacteria and application thereof. The strain C11 is classified and named as Chaetomium globosum (Chaetomium globosum), and is preserved in China center for type culture Collection (CCTCC M2022257) in 2022 and 3 months and 14 days. The strain C11 has antagonism effects on fusarium wilt and verticillium wilt of cotton to different degrees, has promotion effects on cotton growth, and has good development and application prospects.

Description

Strain C11 against plant pathogenic bacteria and its application

technical field

The technical field of biocontrol microorganisms of the present invention particularly relates to strain C11 which antagonizes plant pathogenic bacteria and its application.

Background technique

Cotton is a plant of the Malvaceae family, native to subtropical regions. In the process of planting cotton, there are many common diseases, among which cotton blight and Verticillium wilt are more serious. Cotton verticillium wilt is especially serious at the seedling stage, and it has the title of "cotton cancer". The impact caused by cotton blight and verticillium wilt is even greater, which has a great hindrance to the development of the cotton industry.

At present, in agricultural production, the basic methods for the prevention and treatment of cotton verticillium wilt and cotton wilt include strengthening cultivation management, planting disease-resistant varieties, and chemical control. However, in actual production, these methods have their limitations. For example, the long-term single planting of disease-resistant varieties will reduce their disease resistance; when planting in large areas, it is difficult to accurately control fertilization; Application can also disrupt microbial balance, etc. The use of bio-control bacteria is more beneficial than traditional agriculture in many aspects, and many bio-control bacteria have a greater effect on the growth and promotion of cotton. Therefore, finding more and more efficient biocontrol strains is a technical problem to be continuously solved by those skilled in the art.

Contents of the invention

In view of the above technical problems, the present invention provides strain C11 which antagonizes plant pathogenic bacteria and its application.

In the first aspect of the present invention, the strain C11 that antagonizes plant pathogenic bacteria is classified as Chaetomium globosum, and was deposited in the China Center for Type Culture Collection on March 14, 2022, with the preservation number CCTCC M 2022257.

Further, the plant pathogenic bacteria are Fusarium wilt of cotton, Verticillium dahliae of cotton.

The second aspect of the present invention provides the culture of the strain C11, the culture is a fermentation broth or a volatile metabolite produced during the culture.

The third aspect of the present invention provides a biological agent containing the strain C11 or the culture as an active ingredient.

The fourth aspect of the present invention provides the application of the biological agent in the control of cotton fusarium wilt and cotton verticillium wilt.

The fifth aspect of the present invention provides the application of the coccomella in the prevention and treatment of cotton wilt.

The sixth aspect of the present invention provides the application of the coccoid fungus in the control of cotton verticillium wilt.

The seventh aspect of the present invention provides the application of the biological agent in promoting the growth of cotton.

Further, the indicators of cotton growth include plant height, root length, dry weight of aboveground part, fresh weight of aboveground part, dry weight of underground part and fresh weight of underground part.

The eighth aspect of the present invention provides the application of the said biological agent in increasing the chlorophyll content in cotton.

The present invention has following beneficial effect:

The globular hair fungus of the invention has different degrees of antagonistic effects on cotton sorrel and verticillium dahliae, and has a promoting effect on the growth of cotton.

The biological bacterial agent with the bacterial strain C11 of the present invention as an active ingredient is pollution-free, does not cause environmental pollution, can reduce or even eliminate other corresponding chemical pesticides, not only saves costs, but also improves cotton quality.

Description of drawings

Figure 1 is a picture of the antagonism effect of C11 volatiles on cotton fusarium wilt; B is the front of C11 volatiles on cotton fusarium wilt, A is the front of the control; D is the back of C11 volatiles on cotton fusarium antagonism, and C is the control back.

Figure 2 is the mycelial morphology observation of C11 volatiles antagonized against Fusarium wilt of cotton (scale bar 50mm); B is the mycelial morphology of C11 volatiles antagonized against Fusarium wilt of cotton, and A is the control mycelial morphology.

Fig. 3 is the observation of conidia morphology after C11 volatiles antagonize cotton Fusarium wilt (scale bar 50mm); B is the conidia after C11 volatiles antagonize cotton Fusarium wilt, and A is control mycelial spores.

Fig. 4 is the average number of conidia sizes that C11 volatiles antagonize against Fusarium wilt.

Figure 5 is the effect diagram of backgraft culture after C11 volatiles antagonize cotton Fusarium wilt; B is the positive effect of C11 volatiles on cotton Fusarium wilt antagonism, A is the positive side of the control; D is the reverse side of C11 volatiles antagonistic effect on cotton Fusarium wilt, C is the back side of the control.

Figure 6 is a diagram of the antagonistic effect of C11 volatiles on Verticillium dahliae of cotton; B is the front of the antagonistic effect of C11 volatiles on Verticillium dahliae of cotton, and A is the front of the control; D is the back of the antagonistic effect of C11 volatiles on Verticillium dahliae of cotton, C is the back side of the control.

Figure 7 is the mycelial morphology observation of C11 volatiles antagonizing Verticillium dahliae of cotton (scale bar 50 mm); B is the mycelial morphology of C11 volatiles antagonizing Verticillium dahliae of cotton, and A is the control hyphae morphology.

Fig. 8 is the conidia morphology observation of C11 and XG3 volatiles against Verticillium dahliae of cotton (scale bar 50 mm); B is the conidia of Verticillium dahliae of cotton antagonized by volatiles of C11, and A is the control mycelium spores.

Fig. 9 is the average number of conidia sizes of C11 volatiles against Verticillium dahliae of cotton.

Fig. 10 is the backgraft culture effect diagram of C11 volatile matter to cotton Verticillium dahliae antagonism; B is the positive effect of C11 volatile matter on cotton Verticillium wilt antagonism, A is the control positive; D is the antagonistic effect of C11 volatile matter on cotton Verticillium dahliae Back, C is the control back.

Figure 11 shows the results of the dry weight and fresh weight of the whole plant after the cotton seedlings were treated with the C11 bacterial suspension and the control group.

Figure 12 shows the dry weight and fresh weight results of the aboveground part of the cotton seedlings treated with the C11 bacterial suspension and the control group.

Figure 13 shows the results of the dry weight and fresh weight of the underground part of the cotton seedlings treated with the C11 bacterial suspension and the control group.

Figure 14 shows the plant height and root length results of the cotton seedlings treated with the C11 bacterial suspension and the control group.

Figure 15 is the chlorophyll results of the cotton seedlings treated with the C11 bacterial suspension and the control group.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

Example 1: Isolation and Identification of Trichomonas

1.1 Isolation and purification of soil antagonistic fungi

The cotton variety is Xinluzhong 37. Three different points in the cotton field were selected to take cotton rhizosphere soil samples, and each point selected a healthy cotton rhizosphere soil. Dilution plate method was used to isolate fungi from cotton rhizosphere soil samples. The specific steps are as follows: Take 5g of cotton rhizosphere soil sample, suspend it in 45ml of sterile water, shake and culture it on a horizontal shaker at 80rpm and 25°C for 20min to obtain a suspension, and dilute it to 10-5 times. Using PDA medium, use a pipette to take 0.1 mL of soil dilution solution and spread it on the PDA medium plate, incubate at 25°C for 4-5 days, select well-growing colonies and continue to purify 3 times, observe and record.

1.2 DNA Extraction of Cotton Rhizosphere Soil Microorganisms A rapid DNA extraction method, the specific steps are as follows.

Step 1: Under sterile conditions, use a sterile pipette tip to pick the purified mycelia into a 1.5mL centrifuge tube; Step 2: Add 50uL 10*TE buffer solution, oscillate to disperse the mycelia; Step 3: Seal and microwave 700W for 2min; Step 4: Remove from the ice bath for 2min immediately, and then centrifuge for 10min; Step 5: Pipette slowly to take 10uL of the supernatant as a PCR template.

1.3 Molecular identification of antagonistic fungi

The extracted DNA was used as a template for rDNA-ITS, EF-1α, β-tubulin, and ACT gene sequencing, and Blast comparison was performed on the NCBI website, and the strain with higher sequence homology was selected as a reference strain to evaluate the similarity of species. The primer sequences used are listed in Table 1. The PCR reaction system is 25uL: DNA template 1uL, 2×Es Taq Master Mix 12.5uL, primer-F0.3uL, primer-R0.3uL, ddH2O 10.9uL. The PCR amplification products were detected by 1.5% agarose gel electrophoresis, and then sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing.

Table 1 Primer sequences used and their sources

1.4 Molecular biological identification results of cotton rhizosphere soil fungi

The general primers ITS1 and ITS4 were used to amplify 8 strains, and the fragment lengths obtained were between 570 and 680 bp; the rhizosphere soil fungi were amplified by using EF-1α, β-tubulin and ACT genes respectively, and the amplified fragments ranged from 216 ~229bp, 329~439bp and 268~274bp. The sequencing results were further compared with the gene sequences on NCBI, and the results are shown in Table 2: the ITS sequence of the isolated strain C11 was 100.00% homologous to the ITS sequence of the Chaetomium globosum strain (accession number MN809366.1).

Table 2 Classification information of strain C11

The strain C11 is classified and named as Chaetomium globosum, and was deposited in the China Center for Type Culture Collection (CCTCC) on March 14, 2022. The deposit address is: Wuhan University, Wuhan, China, and the deposit number is CCTCCM 2022257.

Embodiment 2: the application of bacterial strain C11

1. Materials and methods

1.1 Test materials

Pathogens tested: Cotton Fusarium wilt and Cotton Verticillium dahliae were provided by our laboratory and published in: Hao Haiting, Niu Dongdong, Ayi Nisa Ablihaiti, etc. Isolation of fungi from cotton rhizosphere soil and screening of antagonistic bacteria of turmeric pathogen[ J]. Journal of Tarim University. 2020, 32(4).

Endophytic bacteria tested: Chaetomium globosum C11.

Cotton variety: Xinluzhong 37.

1.2 Medium

PDA solid medium (g/L): potato 200g/L, glucose 20g/L, agar 15g/L, water 1L PDA liquid medium (g/L): potato 200g/L, glucose 20g/L , water 1L,

1.3 Antagonistic experiment of strain C11 volatiles against cotton Fusarium wilt

In this experiment, a flat plate with a partition was used for antagonism treatment. A puncher with a diameter of 2.5 mm was used to punch out the bacterial cake of cotton wilt pathogen and inoculate the bacterial cake to one side of the petri dish filled with PDA medium, and the temperature was 26 The incubator at ℃ was cultured upside down for three days, and on the third day, the strain C11 was inoculated to the other side of the petri dish equidistant from the pathogenic bacteria using a puncher of the same diameter. Each treatment was repeated three times, and the PDA medium was used as the control group. Cultivate at 26°C, and observe the antagonistic effect when the pathogenic bacteria in the control group are overgrown.

1.4 Antagonistic experiment of strain C11 volatiles against cotton Verticillium dahliae

In this experiment, a flat plate with a partition was used for confrontation treatment. A puncher with a diameter of 2.5 mm was used to punch out the bacterial cake of Verticillium dahliae and inoculate the bacterial cake to one side of the petri dish filled with PDA medium. The incubator at 26°C was cultured upside down for three days, and on the third day, strain C11 was inoculated on the other side of a petri dish equidistant from the pathogenic bacteria using a hole puncher of the same diameter. Each treatment was repeated three times, and PDA medium was used as the control group . Cultivate at 26°C, and observe the antagonistic effect when the pathogenic bacteria in the control group are overgrown.

1.5 Morphological observation and back-inoculation culture of strain C11 antagonizing Cotton Fusarium and Verticillium dahliae

Use an inoculation needle to pick a small amount of hyphae beside the mycelia to make a temporary slide, and observe the colony shape and color under a microscope; after making a temporary slide with sterile water, observe its large conidia and small conidia under an optical microscope. Conidia, hyphae were morphologically characterized, and pictures were taken.

Using the traditional inoculation method, inoculate the fungus cakes of cotton Fusarium wilt and Verticillium dahliae that have been inhibited by beneficial bacteria into the center of the culture medium and culture them upside down at 26°C for 6 days. Through the growth rate of the colony, observe the effect of the three strains of Trichochleum on Cotton blight, Verticillium dahliae whether to inhibit or kill.

1.6 Growth-promoting effect of cotton

The growth-promoting experiment was carried out using the indoor pot experiment method, setting C11 treatment and a clean water control, each repeated 3 times, and planted 3 to 4 cotton plants in each pot. Place the potted plants in an artificial climate incubator, set the appropriate temperature and humidity, and after 6 to 7 days, the cotyledons grow out of the cotton, prepare the strain C11 bacterial suspension, and use the root irrigation method to pour 5ml of the bacterial liquid on each cotton seedling, 4 After ~5 days, repeat the watering of the bacterial suspension once. After growing for a period of time, the cotton plants were measured, including root length, plant height, chlorophyll content and dry weight and fresh weight of aboveground and underground parts.

2 Results and Analysis

2.1 Antagonism of strain C11 volatiles against Fusarium wilt of cotton, inoculation culture and morphological observation

Using the in-dish antagonism experiment of volatiles against cotton fusarium wilt, through the comparison of the antagonism effect between the treatment group and the control group, the volatiles of the strain C11 have antagonism effect on cotton fusarium wilt, and the cotton fusarium wilt did not spread to the other side of the dish, as shown in the figure 1.

By observing the morphology of mycelia, the hyphae of the control group grew intact, and there were no phenomena such as cavities, fractures and dissolutions, while the pathogenic bacteria treated with strain C11 appeared dissolution, cavities and fractures, as shown in Figure 2; The spore morphology of the pathogenic bacteria, and the changes in the size of the treated conidia of the pathogenic bacteria and the control group illustrate the antagonistic effect. It can be seen that the spores after the treatment of the bacterial strain C11 are smaller than the control group, as shown in Figure 3 and Figure 4.

The treated pathogenic bacteria were inoculated by the traditional inoculation method, and it was observed that the strain C11 inhibited the growth of the cotton wilt pathogen, but did not kill the pathogenic bacteria, as shown in Figure 5.

2.2 Antagonism of strain C11 volatiles against Verticillium dahliae in cotton, inoculation culture and morphological observation

Using the in-dish antagonism experiment of volatiles against Verticillium dahliae of cotton, through the comparison of the antagonism effect between the treatment group and the control group, the volatiles of the strain C11 had a significant antagonism effect on Verticillium dahliae of cotton, and the cotton fusarium wilt did not spread to the other side of the dish. Figure 6.

By observing the morphology of mycelia, the hyphae in the control group grew intact, and there were no phenomena such as cavities, fractures, and dissolution, while the pathogenic bacteria treated with antagonistic bacteria appeared dissolution, cavities, and fractures, as shown in Figure 7; The spore morphology of the pathogenic bacteria, and the changes in the conidia size of the treated pathogenic bacteria and the control group illustrate the antagonistic effect, as shown in Figure 8 and Figure 9.

The treated pathogenic bacteria were inoculated by the traditional inoculation method, and it was observed that the strain C11 inhibited the growth of the cotton Verticillium dahliae pathogenic bacteria, but did not kill the pathogenic bacteria, as shown in Figure 10.

2.3 Determination of plant height, root length, dry weight, wet weight and chlorophyll in cotton pot experiments

In the indoor pot experiment, set the strain C11 treatment and clean water control, through the determination and comparison of the plant height, root length, dry weight, wet weight and chlorophyll of the cotton in the experiment, the growth-promoting effect of the bacterial suspension of the strain C11 on cotton seedlings was tested .

Carefully take out the cotton seedlings, gently shake off the soil on the roots, and measure the fresh weight of the whole cotton seedlings on the balance; put the cotton seedlings in the drying box at 100°C, dry for 30 minutes, and measure the whole plant on the balance Dry weight of cotton seedlings. It can be seen that the fresh weight and dry weight of cotton seedlings irrigated with strain C11 bacterial suspension are higher than those of the control group, as shown in Figure 11.

Take out the cotton seedlings carefully, gently shake off the soil on the roots, and measure the fresh weight of the aboveground part in the balance; put the aboveground part in the drying box and set it at 100 ° C, dry for 30 minutes, and measure the dry weight of the aboveground part in the balance. Heavy. It can be seen that the fresh weight and dry weight of the cotton seedlings irrigated with the strain C11 bacterial suspension are higher than those of the control group, as shown in Figure 12.

Take out the cotton seedlings carefully, shake off the soil on the roots gently, and measure the fresh weight of the underground part in the balance; put the underground part in a drying box and set it at 100 ° C, dry for 30 minutes, and measure the dry weight of the underground part in the balance. Heavy. It can be seen that the dry weight and fresh weight of the underground part of the cotton seedlings treated with the bacterial suspension of strain C11 are both reduced compared with the control group, as shown in Figure 13.

Use a ruler to measure the plant height of the cotton seedlings and record them; carefully take out the cotton seedlings, gently shake off the soil at the roots, measure with a ruler, and record the root length. It can be seen that the plant height and root length of the cotton seedlings treated with the bacterial suspension of strain C11 increased compared with the control group, as shown in Figure 14.

Weigh 0.1g of cotton leaves, put them into a test tube containing 5ml of 80% acetone after washing, shake well, place in the dark at room temperature for 48 hours, absorb 2ml of the extract, add 2ml of 80% acetone to dilute, and use 80% acetone as a blank control , Measure the absorbance at 663.2nm and 646.8nm, and record as shown in Figure 15. It can be seen that at 663.2nm and 646.8nm, the chlorophyll content measured in the cotton seedlings treated with the bacterial suspension of strain C11 is higher than that of the control group.

While preferred embodiments of the invention have been described, additional changes and modifications to these embodiments can be made by those skilled in the art once the basic inventive concept is appreciated. Therefore, it is intended that the appended claims be construed to cover the preferred embodiment as well as all changes and modifications which fall within the scope of the invention.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

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Claims (6)

1. The strain C11 that antagonizes plant pathogenic bacteria, classified as Chaetomium globosum , was deposited in the China Center for Type Culture Collection on March 14, 2022, and the preservation number is CCTCC NO.M 2022257. 2. The biological bacterial agent taking the bacterial strain C11 described in claim 1 as an active ingredient. 3. the application of biological bacterial agent described in claim 2 in the control cotton wilt. 4. the application of biological bacterial agent described in claim 2 in the prevention and treatment of cotton verticillium wilt. 5. the application of biological bacterial agent described in claim 2 in promoting the growth of cotton. 6. the application of the biological bacterial agent described in claim 2 in improving the chlorophyll content in the cotton body.

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