CN111869682B

CN111869682B - Application of chaetomium globosum in preventing and treating wheat take-all

Info

Publication number: CN111869682B
Application number: CN202010832745.XA
Authority: CN
Inventors: 徐飞; 宋玉立; 王俊美; 张姣姣; 韩自行; 李亚红; 李丽娟; 刘露露
Original assignee: Institute of Plant Protection of Henan Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Henan Academy of Agricultural Sciences
Priority date: 2020-08-18
Filing date: 2020-08-18
Publication date: 2021-12-21
Anticipated expiration: 2040-08-18
Also published as: CN111869682A

Abstract

The invention provides an application of chaetomium globosum in preventing and treating wheat take-all, wherein the preservation name of the chaetomium globosum is 12XP1-2-3, the preservation number is CGMCC No.17183, the preservation unit is China general microbiological culture Collection center, and the preservation date is 2019, 1 month and 23 days. The inhibition rate of the Chaetomium globosum 12XP1-2-3 on a flat plate to wheat take-all pathogen GaNS90 is 45.4%, and field test results show that the Chaetomium globosum 12XP1-2-3 is used for coating wheat seeds, so that the wheat white spike rate and the disease index can be obviously reduced.

Description

Application of chaetomium globosum in preventing and treating wheat take-all

Technical Field

The invention relates to an application of chaetomium globosum in preventing and treating wheat take-all.

Background

Take-all disease is an important root disease of wheat, can cause wheat plants to cluster or die in large pieces, and reduces effective ear number, ear grain number and thousand grain weight. The wheat can be infected in seedling stage and adult stage. The typical symptom is that the root and stem base of the wheat are blackened, also called black foot disease, and flaky white spikes appear in the field in the filling stage.

The control is carried out by using control measures such as seed dressing with chemical agents for a long time, but the long-term and excessive use of the chemical agents not only damages the soil micro-ecological environment and aggravates the environmental pollution, but also easily delays the wheat emergence period and reduces the emergence rate. Therefore, the emphasis of disease control is gradually shifting to biological control and agricultural control measures. In the field of crop plant protection, soil microorganisms with obvious biocontrol effect on pathogenic bacteria are screened by utilizing healthy plant rhizosphere soil of crops, and a microbial agent is prepared, so that the method is one of important means for biological control research and is also an important way for developing and utilizing beneficial microbial resources. Chaetomium sp fungi are widely distributed in soil and in plants. The microorganisms can produce various antibiotics such as chaetomium, chaetomium globosum and the like, and are widely researched as biological control bacteria of plant pathogenic bacteria. At present, the separated Chaetomium globosum is not reported to prevent and treat wheat take-all.

Disclosure of Invention

The invention provides an application of chaetomium globosum in preventing and treating wheat take-all, the inhibition rate of the chaetomium globosum 12XP1-2-3 on a flat plate to wheat take-all pathogen GaNS90 is 45.4%, and the field efficacy test result shows that the chaetomium globosum 12XP1-2-3 can reduce the wheat spikelet rate and the disease level and disease index after coating wheat seeds.

The invention provides an application of chaetomium globosum in preventing and treating wheat take-all;

the preservation name of the chaetomium globosum is 12XP1-2-3, the preservation number is CGMCC number 17183, the preservation unit is the China general microbiological culture Collection center of the culture Collection of microorganisms, and the preservation date is 2019, 1 month and 23 days.

The invention also provides application of the biocontrol microbial inoculum containing the chaetomium globosum in preventing and treating wheat take-all.

The biocontrol microbial inoculum is prepared by the following method: and carrying out propagation culture on the chaetomium globosum strain to obtain a spore suspension, and dispersing the spore suspension into a sodium carboxymethylcellulose solution to obtain the biocontrol microbial inoculum.

Preferably, the mass fraction of the sodium carboxymethyl cellulose solution is 4%, and the volume ratio of the spore suspension to the sodium carboxymethyl cellulose solution is 3: 1.

When the biocontrol microbial inoculum is specifically applied, the coating concentration is 5.0-5.3 multiplied by 10⁴The wheat seeds were coated with ascospores/seed.

Biological preservation Instructions

Biological material: chaetomium globosum 12XP 1-2-3; and (3) classification and naming: chaetomium globosum (Latin name: Chaetomium globosum) is preserved in the China general microbiological culture Collection center in 2019, 23.01.23.The addresses of the preservation center are as follows: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation number is CGMCC No. 17183.

Compared with the prior art, the invention has the beneficial effects that:

the Chaetomium globosum 12XP1-2-3 provided by the invention has the inhibition rate of 45.4% on a flat plate against wheat take-all pathogen GaNS90, and the results of indoor pot experiments show that the Chaetomium globosum 12XP1-2-3 can obviously reduce the disease index of wheat and increase the plant height of wheat plants; the field efficacy test result shows that after the wheat seeds are coated by using the chaetomium globosum 12XP1-2-3, the white spike rate caused by the wheat take-all disease is reduced by 32.9 percent, and the disease level and the disease index can also be reduced.

Drawings

FIG. 1 shows the plate antagonistic effect of Chaetomium globosum 12XP1-2-3 on GaNS 90.

Detailed Description

The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative and not limiting of the scope of the invention.

Example 1

Isolation of the Chaetomium globosum strain 12XP1-2-3

In the filling period of wheat, collecting plants which are not attacked in the wheat take-all disease field in the west county of Henan province and bagging. The samples were stored in a refrigerator at 4 ℃. Cleaning the root of a sample, taking 2-3cm of wheat root tissue, sterilizing the surface of the wheat root tissue for 10-30s by using 75% of absolute ethyl alcohol by mass fraction, sterilizing the wheat root tissue for 1.5min by using 1% of NaCIO by mass fraction, washing the wheat root tissue for 3 times by using sterile water, cutting the wheat root tissue into small sections of about 0.5cm after absorbing water by using sterile filter paper, placing the small sections on a potato glucose agar culture medium (PDA, the raw materials comprise 300g of peeled potato, 20g of glucose, 20g of agar and 1000ml of distilled water) flat plate by using sterile tweezers, culturing the small sections at 20 ℃ for 5-7 days, picking edge hyphae to grow on a new PDA flat plate, and after 5-7 days under the same condition, picking out hyphae blocks with uniform bacterial colonies, placing the hyphae blocks on a PDA inclined plane for culturing, and storing the hyphae blocks at 4 ℃.

Example 2

DNA extraction of Chaetomium globosum strain 12XP1-2-3

The chaetomium globosum strain 12XP1-2-3 is picked up on a PDA culture medium, cultured for 5-7 days at 20 ℃, and 5 marginal mycelium blocks are picked up and evenly placed on a PDA plate paved with sterilized glass paper. After 3-5 days, the mycelium was scraped with a small sterilized iron spatula, snap frozen with liquid nitrogen, and stored in a freezer at-20 ℃. When extracting DNA, taking out 20-25 mg hypha and putting into a precooled EP tube with 1.5 ml; adding a little liquid nitrogen, and grinding hyphae into powder in an EP tube by using a precooled iron nail; add 500. mu.l Extraction Buffer (50mM Tris-Cl (pH 8.0), 150mM NaCl, 100mM EDTA (8.0)), shake the suspension pellet on a vortex shaker and mix; then adding preheated 25 mul (mass fraction is 20 percent) of sodium dodecyl sulfate, reversing and mixing evenly, and placing in a water bath kettle at 37 ℃ for 1-3 hours; then adding 75 μ l of 5M NaCl, reversing and mixing; adding 65 μ l CTAB/NaCl (10% CTAB, 0.7M NaCl) solution, and water-bathing at 65 deg.C for 30 min; equal volumes (700. mu.l) of Tris-phenol were added: chloroform: a mixture of isoamyl alcohol (25:24:1) was mixed, centrifuged at 10000rpm at 4 ℃ for 10 minutes, and the supernatant (550. mu.l) was transferred to another EP tube; then adding 0.6 times volume of precooled isopropanol (330 μ l), precipitating at 20 ℃ for 10 minutes, centrifuging at 10000rpm and 4 ℃ for 10 minutes, and removing the supernatant; washing the precipitate with 70% ethanol twice, and air drying in a workbench for 5-10 min; the dried precipitate was dissolved in a suitable amount of sterile ddH₂Storing at-20 deg.C in O.

Example 3

Molecular identification of chaetomium globosum strain 12XP1-2-3

The test uses primers ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') to amplify DNA fragments containing ITS1, 5.8S rDNA and ITS2 of the test strain. Systems are referenced to Daval et al (2010). And (3) amplification reaction conditions: the PCR reaction program is: 3min at 95 ℃; 45s at 95 ℃, 30s at 50 ℃, 1min at 72 ℃ and 35 cycles; 10min at 72 ℃. After sequencing the PCR products, BLAST sequence alignment analysis was performed in NCBI. The sequence alignment result is the biocontrol bacterium 12XP1-2-3 which is the spherical hair shell.

TABLE 1 PCR reaction System

Composition (I)	Concentration of	Dosage (mu l)
			10 XPCR buffer	10mM	5.0
dNTP mixture	10mM	1.0
			Taq DNA enzyme	5u	0.5
Primer 1	20μM	1.0
			Primer 2	20μM	1.0
DNA template	10-50ng/μl	1.0
			Double distilled water	Pure water	40.5
Total volume (μ l)		50.0

Example 4

Flat plate antagonistic effect of chaetomium globosum 12XP1-2-3 on wheat take-all pathogen

Selecting Chaetomium globosum 12XP1-2-3 with sterile toothpick, activating on PDA under the culture condition of 25 ℃ and 12h illumination and 12h darkness; activating wheat take-all pathogen GaNS90 on 1/5 PDA; the culture condition is 25 ℃, and no light is emitted; when the bacterial colony is close to the edge of the culture dish (after 7 days), the bacterial colony edges of chaetomium globosum and holotrichia are respectively beaten into a plurality of bacterial cakes by a puncher with the inner diameter of 0.5 cm. Marking two sections of 1/5PDA culture dishes at a distance of 2.5cm from the circle center; respectively putting chaetomium globosum and wheat take-all pathogen fungus cakes at the marked positions, wherein the distance between the centers of the chaetomium globosum and the wheat take-all pathogen fungus cakes is 5 cm; only wheat take-all pathogen bacterial cake is placed in the blank control, the culture condition is 25 ℃, and no illumination is provided. The growth of wheat take-all pathogen was determined by streaking after 10 days, and the results are shown in FIG. 1. The result shows that the inhibition rate of Chaetomium globosum 12XP1-2-3 on the flat plate to wheat take-all pathogen GaNS90 is 45.4%.

Example 5

Prevention and treatment effect of chaetomium globosum 12XP1-2-3 on indoor potted plants

Taking enough wheat seeds of 'Zhoumai 18', cleaning, sterilizing with sodium hypochlorite with the mass fraction of 3% for 1-3min, and soaking in clear water for 24h for later use. The wheat take-all bacterial strain is activated on PDA, then marginal hypha blocks are picked and cultured on 1/5PDA plates (20ml) for 7 days, and the bacterial strain is inoculated for standby. Chaetomium globosum 12XP1-2-3 is activated to grow on PDA culture medium for 10 days for later use. And (3) beating the cultured chaetomium globosum and the holotrichia into a plurality of fungus cakes by using a puncher with the inner diameter of 9mm respectively. The resistance of Chaetomium globosum 12XP1-2-3 to take all over the disease was determined by a glass tube (18 cm in length and 2.5cm in diameter) method. The bottom of each test tube is provided with a hole for watering. Each test tube is sequentially added with cotton balls, 6cm deep sterilized vermiculite, 2 9mm holomyces blocks, 1cm deep vermiculite, 2 9mm chaetomium globosum mycelium blocks, 1cm deep vermiculite, 3 wheat seeds and 1cm vermiculite from bottom to top. Each 9mm hypha block is divided into 4 small blocks on average by a picking needle. After the seeds are planted, 10ml of distilled water is poured into each tube, the mouth of each glass tube is sealed by a preservative film, and the glass tubes are moved into an incubator at 25 ℃ for germination. After two days, the culture medium is transferred into an incubator with 16 ℃, 12h illumination and 90% humidity. Each tube was dosed weekly with 10ml (1/3v/v) of Hoagland's macronutrient solution. Disease investigation was performed according to the following classification criteria. The root cortex or the central column is browned or blacked to be the disease root. After four weeks of inoculation, the wheat seedlings were pulled out, vermiculite was washed away, disease grade was recorded and plant height, dry root weight, dry stem leaf weight were measured.

The classification standard is as follows:

0 is healthy; the area of the black root accounts for less than 10 percent of the total root area; 2, the area of the black root accounts for 10 to 25 percent of the total root area; the area of the black root accounts for 25 to 50 percent of the total root area; the area of the black root accounts for 50-100% of the total root area; 5-the roots of all wheat seedlings blackened and extended to the base of the stem; 6 ═ plaques spread to the stem; 7-plants fade and stop growing; the 8 ═ plant died.

The results are shown in Table 2.

TABLE 2 prevention and treatment effects of Chaetomium globosum 12XP1-2-3 potted in house on Zhoumai 18

Treatment of	Index of disease condition	Plant height (cm)	Dry weight of individual plant
				12XP1-2-3 seed coating	0.46b	24.8a	0.085a
Seed is not coated	0.76a	19.3b	0.071a
				LSD(p<0.05)	0.217	3.77	0.026

Note: the same letters indicate no significant difference at the 0.05 level, and different letters indicate significant differences at the 0.05 level.

As shown in Table 2, the Chaetomium globosum 12XP1-2-3 can obviously reduce the disease index of wheat and increase the plant height by coating wheat seeds.

Example 6

Chaetomium globosum 12XP1-2-3 seed coating field effect for preventing and treating wheat take-all disease

1. Field control effect of chaetomium globosum on wheat take-all disease

1.1 culture of wheat take-all inoculum

Soaking semen Tritici Aestivi for 12h, sterilizing for 60min, standing for 2 days, sterilizing for 30-60 min, and cooling. Selecting a wheat holopathogenic bacterium strong pathogenic strain KX-7 and a medium pathogenic strain GaNS-90 on a PDA flat plate containing antibiotics, growing for 7 days, selecting edge hyphae to grow on a fresh PDA flat plate, inoculating hypha blocks into a sterilized wheat grain polyethylene bag after 7 days, growing for 3 weeks at room temperature until the hyphae blocks become black, mixing the wheat grains cultured by the strong pathogenic strain KX-7 and the medium pathogenic strain GaNS-90 in equal amount, air-drying, and storing in a refrigerator at 4 ℃.

1.2, building a field disease nursery

And constructing a disease nursery according to the ratio of the seed quantity to the quality of the inoculum of 3: 1. Inoculating 5cm below the seeds, sowing wheat holorust germ and wheat grain culture in the soil by about 10cm, and sowing chaetomium globosum coated and holorust coated seeds in the soil by 5 cm.

1.3 preparation of Chaetomium globosum seed coating

Activating the selected Chaetomium globosum strain on fresh PDA, then respectively selecting mycelium blocks to 100 PDA culture dishes with the length of 9cm, adding sterile water after 15 days, grinding the ascospore by using a coating rod, controlling the ascospore suspension to be about 100mL, and controlling the concentration to be 1.0-2.0X 10⁷Per mL; if the concentration is too low, centrifuge at 3000 rpm for 5min, then remove water and resuspend. 75mL of bacterial liquid (1.3X 10)⁷one/mL) was added 25mL of a solution of 4% by mass of a carboxymethyl cellulose salt to obtain 100mL of a 1% carboxymethyl cellulose salt suspension (1.0X 10)⁷one/mL). 100mL 1X 10⁷Adding 5kg of wheat seeds into 1% of bacterial liquid of carboxymethyl cellulose salt per mL, and uniformly mixing. The spore number on the seed surface is 5.0-5.3 multiplied by 10 after the chaetomium globosum 12XP1-2-3 coated seeds are washed and coated on a flat plate⁴Ascospores per seed. The 2016-year-old 2017 wheat variety is 'Zhengmai 101', the 2017-year-old 2018 wheat variety is 'Bainong 207', and the contrast group medicament is 125g/L of holothurin 10mL/5kg of coated seeds.

1.4 data investigation and recording

The field sample sampling method comprises the following steps: investigating the rate of emergence in the seedling stage, and measuring the plant height, the main root length, the fresh weight, the dry weight and the colonization capacity of chaetomium globosum; the disease incidence is investigated at the tillering stage, and the plant height, main root length, fresh weight, dry weight, tillering number and the colonization capacity of chaetomium globosum are measured; investigating morbidity in the jointing stage; investigating the white spike rate in the grouting period, investigating the 9m double-row white spike number and the total spike number for each cell in an investigation mode, calculating to obtain the white spike rate of the cell, sampling and selecting 30 stems at five points in each cell, taking 270 stems in total for each treatment back to a laboratory, washing the stems under a water tap, and determining the disease grade, wherein the grading standard of the adult plant stage is as follows:

0 is no disease root; the disease area of the root system is less than 10 percent; 2, the disease area of the root system accounts for 11 to 25 percent; the disease area of the root system accounts for 26-50%; 4, the disease area of the root system accounts for 51 to 75 percent; 5, the disease area of the root system accounts for 76 to 100 percent or the base of the stem is blackened. The results are shown in Table 3.

Surface 32016 and 2017 wheat chaetomium globosum seed coating effects on preventing and treating wheat take-all disease in wheat filling stage

As can be seen from Table 2, investigation of seedling stage and tillering stage shows that the wheat is rarely infected by holotrichia, investigation of filling stage shows that the 2016 + 2017 year wheat variety is 'Zheng wheat 101', the white ear rate of the blank control group is 7.3%, and the white ear rate of the chaetomium globosum 12XP1-2-3 seed coating treatment is 4.9%. The wheat variety in 2017 and 2018 years is Bainong 207, the white ear rate of the blank control group is 9.9 percent, and the white ear rate of the chaetomium globosum 12XP1-2-3 seed coating treatment is 3.9 percent. The suggestion is that the chaetomium globosum 12XP1-2-3 provided by the invention can reduce the white spike rate and the disease level and disease index after coating the seeds.

It should be noted that the steps and methods adopted in the claims of the present invention are the same as those of the above-mentioned embodiments, and for the sake of avoiding redundancy, the present invention describes the preferred embodiments, but those skilled in the art can make other changes and modifications to these embodiments once they learn the basic inventive concept. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. The application of chaetomium globosum in preventing and treating wheat take-all is caused by a take-all strain GaNS90 and/or a strain KX-7;

the preservation name of the chaetomium globosum is 12XP1-2-3, the preservation number is CGMCC No.17183, the preservation unit is China general microbiological culture Collection center, and the preservation date is 2019, 1 month and 23 days.

2. The application of the biocontrol microbial inoculum containing the chaetomium globosum in the prevention and treatment of the wheat take-all.

3. The use of claim 2, wherein the biocontrol microbial inoculum is prepared by the following method: and carrying out propagation culture on the chaetomium globosum strain to obtain a spore suspension, and dispersing the spore suspension into a sodium carboxymethylcellulose solution to obtain the biocontrol microbial inoculum.

4. The use according to claim 3, wherein the mass fraction of the sodium carboxymethyl cellulose solution is 4%, and the volume ratio of the spore suspension to the sodium carboxymethyl cellulose solution is 3: 1.

5. Use according to any one of claims 2 to 4, characterized in that the coating concentration is 5.0 to 5.3 x 10⁴Coating treatment of wheat seeds with ascospore/seed。