CN112868675A

CN112868675A - Method for preventing and treating clubroot of Chinese cabbage by combining ozone water and trichoderma

Info

Publication number: CN112868675A
Application number: CN202110377096.3A
Authority: CN
Inventors: 魏艳丽; 李纪顺; 李红梅; 扈进冬; 赵忠娟; 陈凯; 杨合同
Original assignee: Ecology Institute Of Shandong Academy Of Sciences (the Sino-Japanese Friendship Biotechnology Research Center Shandong Academy Of Sciences)
Current assignee: Ecology Institute Of Shandong Academy Of Sciences (the Sino-Japanese Friendship Biotechnology Research Center Shandong Academy Of Sciences)
Priority date: 2021-04-08
Filing date: 2021-04-08
Publication date: 2021-06-01
Anticipated expiration: 2041-04-08
Also published as: CN112868675B

Abstract

The invention relates to the technical field of biological control of plant diseases, in particular to a method for controlling clubroot of Chinese cabbage by combining ozone water and trichoderma, which comprises the steps of (1) irrigating soil by using ozone water with the concentration of 6-10 mg/L, and irrigating 70-100 m of ozone water in each mu of soil³Ozone water; (2) when the water content of the soil is naturally evaporated and reduced to be capable of mechanically preparing soil, applying the trichoderma harzianum preparation as a base fertilizer, wherein the effective viable count of the trichoderma harzianum preparation applied to each mu of soil is (0.5-5) multiplied by 10¹²A CFU; (3) before sowing, dressing seeds of Chinese cabbage with the trichoderma atroviride preparation, dressing seeds of every 150g of Chinese cabbage with the trichoderma atroviride preparation, wherein the effective viable count of the trichoderma atroviride preparation is (1-10) x 10⁸CFU/g. The method of the invention is rightThe prevention and treatment effect of the clubroot of the Chinese cabbage can reach more than 90 percent, and the micro-ecological environment of the soil is not influenced.

Description

Method for preventing and treating clubroot of Chinese cabbage by combining ozone water and trichoderma

Technical Field

The invention relates to the technical field of biological control of plant diseases, in particular to a method for controlling Chinese cabbage clubroot by combining ozone water and trichoderma.

Background

Chinese cabbage clubroot is prepared from radical clubroot fungusPlasmodiophora brassicaeWoron.) infection, a soil-borne disease. The disease damages the roots of the Chinese cabbages, and the roots are rotten or even withered to death in severe cases. Under the influence of a plurality of factors such as soil acidification, climate warming, increase of cultivation area and multiple cropping index, clubroot has a tendency of increasing year by year, cruciferous crops such as Chinese cabbage with 320-.

At present, the Chinese cabbage clubroot is mainly controlled by taking measures of spraying chemical pesticide, spraying lime to improve the physical and chemical properties of soil, selecting suitable disease-resistant varieties and the like. Part of chemical agents are unstable in control effect and easy to cause environmental pollution, and soil hardening and alkalization are easy to cause by long-term application of quicklime. The green prevention and control technical regulation of the clubroot of the Chinese cabbage is sought and established, and the safe and efficient prevention and control of the clubroot have important significance for the Chinese cabbage planting industry.

Ozone is a gas with strong oxidizing property, is very easy to dissolve in water, can destroy the structure of a microbial film through the oxidizing action, has extremely strong biocidal oxidizing action, not only inhibits the survival of pathogenic bacteria, but also has negative effects on beneficial microorganisms and plant growth of non-target soil, and limits the popularization and application of ozone in the aspect of agricultural plant diseases and insect pests.

Disclosure of Invention

Aiming at the technical problem that the extremely strong biocidal oxidation effect of ozone easily affects the growth of beneficial microorganisms and plants in non-target soil, the invention provides a method for preventing and treating cabbage clubroot by combining ozone water and trichoderma, wherein the ozone water is used for irrigating the soil before planting the cabbage, then the trichoderma harzianum preparation is used as a base fertilizer, and the trichoderma atroviride preparation is used for dressing the cabbage seeds before sowing, so that the prevention and treatment effect on the cabbage clubroot can reach more than 90%, and the micro-ecological environment of the soil is not affected.

A method for preventing and treating Chinese cabbage clubroot by combining ozone water and trichoderma comprises the following steps:

(1) irrigating the soil with 6-10 mg/L ozone water, wherein the water is 70-100 m per mu of soil³Ozone water;

(2) when the water content of the soil is naturally evaporated and reduced to be capable of mechanically preparing soil, applying the trichoderma harzianum preparation as a base fertilizer, wherein the effective viable count of the trichoderma harzianum preparation applied to each mu of soil is (0.5-5) multiplied by 10¹²CFU；

(3) Before sowing, dressing seeds of Chinese cabbage with the trichoderma atroviride preparation, dressing seeds of every 150g of Chinese cabbage with the trichoderma atroviride preparation, wherein the effective viable count of the trichoderma atroviride preparation is (1-10) x 10⁸CFU/g。

Further, in the step (1), determining the concentration of ozone water according to the base number of plasmodiophora brassicae in the soil, wherein the base number of plasmodiophora brassicae in the soil is more than 10⁶When CFU/g is needed, the concentration of ozone water is selected to be 8-10 mg/L; less than or equal to 10⁶And when CFU/g is adopted, the concentration of ozone water is selected to be 6-8 mg/L.

Further, in the step (1), the determination method of the plasmodiophora brassicae base number in the soil comprises the following steps: extracting soil sample DNA, and extracting with Leptoma brassicae (Leptoma brassicae) (II)Plasmodiophora brassicaeWoron.) specific primers PBF (5'-GAACGGGTTCACAGACTAGAT-3') and PBR (5'-GCCCACTGTGTTAATGATCC-3') were subjected to qPCR amplification and the number of clubroot cells in the soil was calculated.

Further, in the step (1), in order to ensure effective ozone concentration, the ozone water is irrigated in multiple points, for example, one irrigation point is arranged every 10-15 m.

Further, in step (2), the Trichoderma harzianum preparation is Trichoderma harzianum TW21990 preparation, Trichoderma harzianum TW21990 has been deposited in the China general microbiological culture Collection center, depository, at 9.1.2016The address is No. 3 of Xilu No.1 of Beijing Korean-yang district, the preservation number is CGMCC No.12864, and the classification name is Trichoderma harzianumTrichoderma harzianum。

Further, in the step (2), the preparation method of the trichoderma harzianum preparation comprises the steps of preparing conidia by fermenting trichoderma harzianum, and adding turfy soil and/or medical stone to prepare the trichoderma harzianum preparation with the effective viable count of (1-10) multiplied by 10⁸CFU/g of Trichoderma harzianum preparation.

Further, the step (2) further comprises applying 500-1000 kg of mushroom residues and/or 200kg of bio-organic fertilizer to the soil.

Further, in the step (3), the trichoderma atroviride preparation is trichoderma atroviride HB20111 preparation, the trichoderma atroviride HB20111 is stored in the common microorganism center of China general microbiological culture Collection center in 12 months and 5 days in 2018, the storage address is No. 3 of Xilu No.1 of Beijing Kogyo area, the storage number is CGMCC No.16963, and the classification name is trichoderma atrovirideTrichoderma atroviride。

Further, in the step (3), the preparation method of the trichoderma atroviride preparation comprises the step of adding 1% of sodium alginate, 2% of sodium carboxymethylcellulose, 0.2% of calcium chloride and trichoderma atroviride by mass into medical stone serving as a carrier to prepare the trichoderma atroviride preparation with the effective viable count of (1-10) x 10⁸CFU/g of Trichoderma atroviride preparation.

The beneficial effect of the invention is that,

according to the technical scheme, aiming at the soil with serious cabbage clubroot, the ozone water is used for irrigating to reduce the base number of brassica clubroot pathogenic bacteria in the soil, and then the trichoderma agent is applied to restore the micro-ecological environment of healthy soil, so that multiple effects of preventing and treating diseases, promoting the growth of the cabbage and improving the soil are achieved.

Drawings

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.

FIG. 1 is a standard curve diagram established from a standard plasmid in example 1;

FIG. 2 is a photograph of the roots of the cabbage of the control group of example 4;

FIG. 3 is a photograph of roots of the ozone-treated cabbage of example 4;

FIG. 4 is a graph comparing the incidence of clubroot in the cabbages of the different treatment groups of example 4;

FIG. 5 is a graph comparing plant height and dry weight of Chinese cabbage of different treatment groups of example 4;

FIG. 6 is a comparison graph of the number of pathogenic bacteria in the rhizosphere soil of the cabbages of the different treatment groups of example 4.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The Trichoderma harzianum TW21990 preparation used in the following examples was a mixture of conidia prepared by fermentation of Trichoderma and Maifanitum; the trichoderma atroviride HB20111 preparation is a mixture of medical stone, sodium alginate, sodium carboxymethylcellulose, calcium chloride and trichoderma atroviride, wherein the mass percentages of the sodium alginate, the sodium carboxymethylcellulose and the calcium chloride are respectively 1%, 2% and 0.2%.

Example 1 determination of the number of plasmodiophora brassicae bases in soil based on qPCR technology

The rectangular land is sampled by snake shape, and the square land is sampled by five-point cross. The area of the land is less than 670m²Taking 3-5 points in time, which are more than 670m²Taking 6-10 points, and sampling depth is about 20cm of plough layer soil. The soil samples are collected and mixed evenly, the redundant soil is discarded by a quartering method, and 200g of the soil is reserved as a sample. Each plot was sampled repeatedly to obtain 3 samples.

0.5g of Soil sample was weighed, Soil DNA was extracted using DNeasy Power Soil DNA Isolation Kit (Qiagen, Valencia, Calif.) Kit according to the instructions, and DNA quality and concentration were checked by 0.8% agarose gel electrophoresis. Specific primers PBF (5'-GAACGGGTTCACAGACTAGAT-3') and PBR (5'-GCCCACTGTGTTAATGATCC-3') are designed according to an ITS zone conserved sequence of the plasmodiophora brassicae, and the size of an expected amplified fragment is 200 bp. The amplified fragment was ligated to a T-vector to construct a standard plasmid.

A qPCR reaction system was prepared according to the instructions of SYBR Green Premix DimerEraser (Takara, Japan), and a 20. mu.L reaction system consisted of 1 XSSYBR Premix Ex TaqTM 10. mu.L each, 0.4. mu.L each of primers (0.2. mu. mol/L), 2. mu.L of soil DNA, and 7.2. mu.L of ultrapure water. Blank controls replace soil DNA with water.

The apparatus used for the qPCR reaction was iCycler iQ5(Bio-Rad, California, USA), with a reaction condition of pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 10s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 20s, for 40 cycles; after circulation is finished, the sample is heated to 95 ℃, the temperature is immediately reduced to 60 ℃ and kept for 5s, then the temperature is increased by 0.5 ℃ every 5s to be increased to 95 ℃, a melting curve is drawn, a standard curve (figure 1) is established by standard plasmids, and the base number of the plasmodiophora brassicae in the soil can be calculated according to the standard curve.

Example 2 inhibition of Oncorhynchus in soil by ozone water of different concentrations

Weighing 100g of fresh cabbage clubroot tissue, adding 100mL of sterile water, and homogenizing at low temperature to obtain clubroot spore suspension. And measuring the suspension, mixing the suspension with sterilized soil according to the v/w ratio of 5% and 15% respectively to prepare low-content and high-content diseased soil, and reserving a sample for testing. The test pots are circular pots with the depth of 20cm and the diameter of 15cm, 2kg of soil is filled in each pot, 2L of ozone water is poured in each pot by ozone water with the concentration of 2mg/L, 4mg/L, 6mg/L, 8mg/L and 10mg/L respectively, 2L of ozone water is poured in each pot, the same amount of sterile water is poured in a contrast mode, and 3 times of treatment are repeated. After the sterile culture chamber was left for 7 days, 0.5g of Soil sample was mixed per pot, and Soil DNA was extracted using DNeasy Power Soil Isolation Kit (Qiagen, Valencia, CA) according to the instructions, and qPCR was performed using PBF/PBR as primers to calculate the number of Plasmodium falciparum in the Soil, and the results are shown in Table 1.

TABLE 1 content of Plasmodiophora (x 10) in soil treated with ozone water of different concentrations⁵CFU/g）

Different soil samples	Before treatment	Sterile water	2mg/L	4mg/L	6mg/L	8mg/L	10mg/L
								5% low content soil	416.24	234.17	61.38	0.23	0.17	0.02	0.00
25% high content disease soil	1386.36	1081.46	276.35	17.86	0.35	0.11	0.02

The results show that: the quantity of plasmodiophora elata in the soil can be obviously inhibited by irrigating with ozone water. The ozone water treatment with concentration of 4mg/L can increase the amount of plasmodiophora elata in 5% low-content diseased soil from 4.16 × 10⁷CFU/g is reduced to 2.3X 10⁴CFU/g, the number of the plasmodiophora diophora in 25 percent high-content diseased soil is 1.39 multiplied by 10⁸The CFU/g is reduced to 1.79 multiplied by 10⁶CFU/g; the ozone water treatment with concentration of 8mg/L can reduce the amount of plasmodiophora elata in 5% low-content diseased soil to 0.2 × 10⁴CFU/g, the number of the plasmodiophora elata in 25 percent high-content diseased soil is reduced to 1.1 multiplied by 10⁴CFU/g, ozone water with concentration of 10mg/L is used for treatment, and plasmodiophora cannot be basically detected in the two soils.

The spore amount of plasmodiophora brassicae in general soil is more than 10⁴CFU/g, clubroot may occur. Comprehensively considered, when the radical number of the plasmodiophora brassicae in the soil is less than 10⁶When CFU/g is adopted, the concentration of ozone water is 6-8 mg/L; higher than 10⁶The concentration of the ozone water is 8-10 mg/L at CFU/g.

Example 3 influence of ozone water of different concentrations on sprouting and growth of Chinese cabbage

The potting soil is collected in an experimental base of an ecological research institute of academy of sciences of Shandong province, is common garden soil, is removed with 1-2 cm of surface soil, is collected with 0-20 cm of soil sample, is sampled at multiple points and is uniformly mixed, and is sieved by a 50-mesh sieve. The test pots were circular pots 20cm deep and 15cm in diameter, each pot contained 2kg of soil, each pot was irrigated with 2L of ozone water, 4mg/L, 6mg/L, 8mg/L, 10mg/L, each pot was irrigated with 2L of ozone water, the same amount of sterile water was irrigated, 3 replicates of each treatment were set, seeding was carried out when the soil humidity was appropriate, the management was carried out routinely, each growth index of the cabbage was investigated after 4 weeks, and the results are shown in table 2.

TABLE 2 Effect of ozone water treatment on Chinese cabbage seedlings

Treatment of	Germination rate/%	Average plant height/cm	Dry weight per gram of individual plant	Chlorophyll content/(mg/L)
					Control	96.41±2.35a	13.15±1.38a	0.57±0.03a	0.508±0.012a
2mg/L	94.05±1.64a	14.76±0.87a	0.65±0.18a	0.498±0.025a
					4mg/L	97.82±1.91a	13.67±0.63a	0.61±0.10a	0.514±0.023a
6mg/L	96.53±2.62a	10.06±0.56ab	0.55±0.07a	0.528±0.012a
					8mg/L	91.59±1.24a	9.34±0.81b	0.45±0.03b	0.537±0.084a
10mg/L	92.48±2.76a	8.97±0.38b	0.41±0.02b	0.551±0.116b

Note: results are mean ± standard deviation of triplicates, different lower case letters indicate P >0.05 level difference.

The results show that after the ozone water is used for treating the soil, direct sowing is carried out, and the germination rate, the plant height, the single plant dry weight and the chlorophyll content of the Chinese cabbage of the ozone water treatment group with the concentration of less than 4mg/L have no obvious difference with those of a control; however, the ozone water treatment group of 8mg/L has obvious influence on the germination rate of the Chinese cabbage and the growth of seedlings, so that the Chinese cabbage is short and small, and the difference between the plant height and the dry weight of a single plant and other treatments is obvious. The reason is that the strong biocidal oxidation effect of ozone destroys the micro-ecological environment of soil, thereby affecting the growth of plants, and beneficial microorganisms such as trichoderma need to be added after ozone treatment to rebuild the healthy soil micro-ecological environment.

Example 4 prevention and treatment of clubroot of cabbage with combination of ozone water and Trichoderma

Selecting a land block with serious incidence of the cabbage clubroot, removing 1-2 cm of surface soil, collecting a soil sample of 0-20 cm, sampling at multiple points, uniformly mixing, and sieving with a 50-mesh sieve.

The test was set to 4 treatments, Control (CK), ozone treatment (O), trichoderma preparation (T), ozone + trichoderma preparation (OT), each treatment set to 4 replicates. The test pot is a circular flowerpot with the depth of 20cm and the diameter of 15cm, and each pot is filled with 2kg of soil. Then directly irrigating with ozone for ozone treatmentThe ozone water concentration is adjusted to 8mg/L, 2L of ozone water is poured into each basin, and the same amount of tap water is poured into the control basin. After the soil water content is suitable for planting operation, 1.5g of effective viable bacteria with the number of 3 multiplied by 10 are added into each pot of the trichoderma preparation group and the ozone and trichoderma preparation group⁷CFU/g of a preparation of Trichoderma harzianum TW21990, mixed well with top 10cm of soil. Taking 30g Chinese cabbage seed, and using 1g live bacteria count of 3 × 10⁸The CFU/g trichoderma atroviride HB20111 solid microbial inoculum is used for seed dressing, and 15 seeds are sowed in each pot. Pouring water once every 3-4 d according to the dry and wet condition of the soil, after culturing for 5 weeks in a greenhouse, investigating the disease conditions of the Chinese cabbage plant height, dry weight and clubroot, carrying out qPCR reaction on the DNA of the cabbage rhizosphere soil by taking PBF/PBR as a primer, and detecting the quantity of pathogenic bacteria in the rhizosphere soil, wherein the specific detection method is the same as that in example 1.

As shown in fig. 2-5, the roots of the Chinese cabbages in the control treatment group are expanded and deformed, the capillary roots are reduced, and the incidence rate of clubroot is 78.17%; the Chinese cabbage root system is healthy due to the irrigation treatment group with the ozone water, the clubroot disease incidence rate is only 2.5 percent, and the incidence rate of the combined treatment of the ozone water and the trichoderma preparation is 6.25 percent; the influence of ozone water on the plant after soil disinfection can be effectively relieved by trichoderma treatment, and the plant height and the dry weight of the trichoderma treatment are not obviously different from those of a control.

The specific primer qPCR amplification result of plasmodiophora root pathogenic bacteria is shown in figure 6, and compared with a control, the copy number of pathogenic bacteria in rhizosphere soil can be obviously reduced by two treatment groups O and OT irrigated by ozone water. The irrigation with ozone water can reduce the number of plasmodiophora brassicae in the soil, thereby reducing the morbidity of the next batch of Chinese cabbages.

Example 5 field control of Chinese cabbage clubroot with combination of ozone water and Trichoderma

The field test is carried out in the Chinese cabbage planting base of Gaofuzhuang village in Li Chongwan town, Qingdao, Shandong province, and the variety is 'Yi and autumn'. The test field is a cabbage continuous cropping field, and the clubroot disease is severe. The test microbial inoculum is Trichoderma harzianum TW21990 preparation and Trichoderma atroviride HB20111 preparation, and the effective viable count of the two preparations is 3 multiplied by 10⁸CFU/g。

4 treatments are set in the field test, and are respectively treatment 1: trichoderma harzianum TW21990 preparation 5 kg/mu + mushroom dregs 800 kg/mu; and (3) treatment 2: 6mg/L ozone water 100m³5 kg/mu of irrigation/mu of trichoderma harzianum TW21990 preparation and 800 kg/mu of mushroom residue; and (3) treatment: conventional treatment and mushroom residue 800 kg/mu; and (4) treatment: conventional treatment (farmers are used to plant). Mechanically plowing for 20cm in each treatment, and before sowing, dressing seeds with the trichoderma atroviride HB20111 preparation in the treatment 1 and the treatment 2, wherein the specific operation is to dressing seeds with 5g of the trichoderma atroviride HB20111 preparation per 150g of Chinese cabbage seeds. Disease investigation and yield test were performed at 11 months and 15 days of harvest, and the results are shown in table 3.

TABLE 3 field control of cabbage growth and clubroot by different treatments

Treatment of	Index of disease condition	Preventive effect/%)	Weight per kg of individual plant	Average yield per mu/kg	Increase in yield/%)
						1	38.67	53.59	3.92	7056	28.52
2	8.21	90.15	4.37	7866	43.28
						3	56.84	31.79	3.67	6606	20.33
4	83.33	—	3.05	5490	—

The results show that the combined treatment group effect of the ozone water and the trichoderma in 4 treatments is the best, compared with the conventional treatment, the clubroot prevention effect reaches 87.52%, and the mu yield is increased to 43.28%.

Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention.

Sequence listing

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Claims

1. A method for preventing and treating clubroot of Chinese cabbage by combining ozone water and trichoderma is characterized by comprising the following steps:

2. The method of claim 1, wherein in step (1), the ozone water concentration is determined according to the plasmodiophora brassicae base number in the soil, wherein the plasmodiophora brassicae base number in the soil is more than 10⁶When CFU/g is needed, the concentration of ozone water is selected to be 8-10 mg/L; the base number of plasmodiophora brassicae in soil is less than or equal to 10⁶And when CFU/g is adopted, the concentration of ozone water is selected to be 6-8 mg/L.

3. The method of claim 2, wherein in step (1), the number of plasmodiophora brassicae loci in the soil is determined by:

extracting soil sample DNA, performing qPCR amplification by using plasmodiophora brassicae specific primers PBF and PBR, and calculating the base number of plasmodiophora brassicae in soil, wherein the sequence of PBF is 5'-GAACGGGTTCACAGACTAGAT-3', PBR, and the sequence of PBF is 5'-GCCCACTGTGTTAATGATCC-3'.

4. The method according to claim 1, wherein the ozone water is applied by multi-point irrigation in step (1).

5. The method according to claim 1, wherein in the step (2), the Trichoderma harzianum preparation is Trichoderma harzianum TW21990 preparation, Trichoderma harzianum TW21990 has been deposited in China general microbiological culture Collection center at 1.9.2016, with the deposition address of Beijing-oriented Zhongchen Xilu-1 Hospital No. 3, CGMCC No.12864, and classified and named Trichoderma harzianumTrichoderma harzianum。

6. The method according to claim 1, wherein the trichoderma harzianum preparation in the step (2) is prepared by preparing conidia by fermenting trichoderma harzianum, adding turfy soil and/or medical stone to obtain the active viable count of (1-10) × 10⁸CFU/g of Trichoderma harzianum preparation.

7. The method of claim 1, wherein the step (2) further comprises applying 500-1000 kg of mushroom dregs and/or 200kg of bio-organic fertilizer to the soil.

8. The method as claimed in claim 1, wherein in step (3), the trichoderma atroviride preparation is trichoderma atroviride HB20111 preparation, the trichoderma atroviride HB20111 has been preserved in China general microbiological culture Collection center (CGMCC) within 5 months and 12 months in 2018, the preservation address is No. 3 Hospital No.1 Samura in the sunward area of Beijing, the preservation number is CGMCC No.16963, and the classification name is trichoderma atrovirideTrichoderma atroviride。

9. The method of claim 1, wherein in the step (3), the trichoderma atroviride preparation is prepared by adding 1% of sodium alginate, 2% of sodium carboxymethylcellulose, 0.2% of calcium chloride and trichoderma atroviride to the carrier maifanite according to the mass percentage to obtain the trichoderma atroviride preparation with the effective viable count of (1-10) x 10⁸CFU/g of Trichoderma atroviride preparation.