LU502648B1 - Trichoderma-based seed coating agent, preparation method and application thereof - Google Patents

Abstract

The invention discloses a Trichoderma-based seed coating agent, a preparation method and application thereof. The seed coating agent comprises Trichoderma chlamydospore fermentation broth, film forming agent, chitosan oligosaccharide, phenazine-1-carboxylic acid and humic acid; the fermentation broth of Trichoderma chlamydospore contains 13-17% (W/V) film-forming agent, 0.35-0.45% (W/V) chitosan oligosaccharide, 0.5-1.5 ug/ml phenazine-1-carboxylic acid and 0.05-0.1% (W/V) humic acid. The seed coating agent of the invention has the advantages of convenient preparation, easy coating, high efficiency and low toxicity, is relatively safe to the environment, and has good control effect on soil-borne diseases of corn.

Description

DESCRIPTION LU502648

TRICHODERMA-BASED SEED COATING AGENT, PREPARATION

METHOD AND APPLICATION THEREOF

TECHNICAL FIELD

The invention relates to a 7richoderma-based seed coating, a preparation method and application thereof, in particular to a preparation method of a biocontrol Trichoderma biological seed coating agent and an application technology in controlling corn stalk rot, sheath blight and root rot.

BACKGROUND

Trichoderma is a common habitant fungus in soil and plant rhizosphere ecosystem. Trichoderma is internationally recognized as a beneficial microorganism, which mainly controls soil-borne diseases of various crops, including crop seedling disease, root rot and blight, through the mechanisms of nutrition and space competition, resistance, heavy parasitism and induced resistance. Trichoderma not only has the function of preventing and controlling diseases, but also has the functions of promoting plant root growth, improving nutrient utilization efficiency, increasing crop yield and stress resistance, repairing soil, etc. It is a multifunctional microorganism with remarkable disease prevention and yield increase.

In recent years, the occurrence of soil-borne diseases of corn, such as corn stalk rot and sheath blight, has been increasing year by year. In some areas, the incidence rate is 15-20% in normal years and over 40% in severe years, which has caused serious yield loss, and has become the main threat of high quality and high yield in corn producing areas of China. Not only that, the high incidence of corn stalk rot caused serious lodging of corn, which restricted the extension of mechanized production technology of corn. For many years, 7richoderma-based biocontrol agents against plant disease have been mainly used in the form of compost and granules at home and abroad, and there are few reports on the success of Trichoderma-based seed coating agents.

Trichoderma-based seed coating agent created in Biowork, United States has only been reported for successful control of corn seedling diseases ,however , never reported successful to control stalk rot (Fusarium spp., Pythium spp.) and Rhizoctonia solani in the late growth stage of corn.

Due to the long infection period of corn stalk rot and sheath blight, they can infect corn from seedling stage to adult stage, and the symptoms usually occur after the grain filling stage.

Therefore, in the process of biological control, it is required that the biocontrol microbes have the ability to colonize the soil and crop roots for a long time and the function of systematically inducing plant resistance, so as to realize the prevention and control of corn diseases in the later,502648 growth stage after treating seeds with microorganisms.

SUMMARY

Aiming at the defects in the prior art, the invention aims to provide a Trichoderma based-seed coating agent, a preparation method and application thereof, which can be effectively used for controlling corn stalk rot, sheath blight, root rot and the like. The antagonistic Trichoderma strain obtained by screening from a large number of Trichoderma resources has strong competitiveness in the soil, effectively inhibits the reproduction and colonization of root surface pathogens in the soil, and more importantly, the screened strain has systemic induced resistance, that is, Trichoderma colonizes the root system of corn with the germination of seeds, generates a series of elicitors in the root system to induce the systematic expression of defense response genes in the root system, and the lasting period can reach the whole growth period of corn (Wu

Xiao et al., 2015), thus realizing the prevention and treatment of late diseases such as stem rot, sheath blight and the like. Therefore, it is of great significance to develop a biocontrol

Trichoderma-based seed coating agent which can control soil-borne diseases of corn and popularize it in the whole country for green control of corn, and its application prospect is broad.

The purpose of the invention is realized by the following technical scheme: in the first aspect, the invention provides a 7Trichoderma-based seed coating agent, which comprises [Trichoderma chlamydospore fermentation broth, a film forming agent, chitosan oligosaccharide, phenazine-1-carboxylic acid and humic acid;

The fermentation broth of Trichoderma chlamydospore contains 13-17% (W/V) film-forming agent, 0.35-0.45% (W/V) chitosan oligosaccharide, 0.5-1.5 ug/mL phenazine-1-carboxylic acid and 0.05-0.1% (W/V) humic acid.

Preferably, each liter of Trichoderma chlamydospore fermentation broth contains 1.0pg/ml phenazine-1-carboxylic acid and 0.06% (W/V) humic acid.

Preferably, the film forming agent comprises 853 film forming agent.

Preferably, the Trichoderma strain is Trichoderma harzianum SH2303 with the preservation number of CTCCSJ-A-QT 1266.

Preferably, the preparation of the Trichoderma chlamydospore fermentation broth comprises the following steps: inoculating the first-class strain into a Trichoderma liquid fermentation medium with an inoculation amount of 0.5% (V/V) strain seed liquid, and fermenting and culturing at 28°C for 7 days to obtain the Trichoderma chlamydospore fermentation broth;

The strain seed liquid is obtained by culturing Trichoderma strain discs in PD culture medium gg;500648 180 rpm and 28°C for 5 days with the inoculation amount of 3-5 pieces/triangular flask. The

Trichoderm culture discs is taken by punching the culture colony of PDA along the edge.

Preferably, the composition of the Trichoderma liquid fermentation medium comprises: every 1,000 liters of Trichoderma liquid fermentation medium contains 40 kg of corn flour, 400 g of magnesium sulfate, 6.0 g of ferrous sulfate, 2.0 g of manganese sulfate, 1.6 g of zinc sulfate, 3.06 kg of potassium dihydrogen sulfate, 1.13 kg of sodium nitrate, 880 g of ammonium nitrate, 800 g of calcium chloride and the balance of water, and the initial pH is adjusted to 4-5.

The PD medium and 7richoderma liquid fermentation medium of the invention are sterilized at 121°C for 25 min before use.

Preferably, the tank loading amount of the fermentation culture is controlled as follows: 200 L liquid fermentation medium is loaded into a 300 L fermentor.

In the second aspect, the invention provides a preparation method of the seed coating agent, which comprises the following steps: mixing chitosan, phenazine-1-carboxylic acid, humic acid and Trichoderma chlamydospore fermentation broth according to the proportion, and then adding a film forming agent and mixing to obtain the seed coating agent.

In the third aspect, the invention provides an application of the 7richoderma-based seed coating agent as a biocontrol agent in controlling soil-borne diseases of corn.

Preferably, the soil-borne diseases include controlling corn stalk rot, sheath blight, root rot, etc.

In the fourth aspect, the invention provides an application method of the 7richoderma-based seed coating agent in controlling soil-borne diseases of corn, specifically, uniformly mixing the

Trichoderma-based seed coating agent with corn seeds according to the mass ratio of 1:(10-15).

The invention involves 7richoderm strain by the preliminarily screened, re-screened and purifyed, and which then is stored in the slant culture-medium of a glass test tube. The fermentation broth of 7richoderma chlamydospore was produced by liquid fermentation in corn flour culture broth. The fermentation broth containing a large amount of chlamydospore (100 million CFU /mL) was mixed with film-forming agent, chitosan oligosaccharide, phenazine-1-carboxylic acid and humic acid according to the optimal ratio obtained by response surface design to prepare Trichoderma-based seed coating agent, which coated corn seeds (1:10-1:15 W/W) and sowed in greenhouse and field. Compared with the control, the control effect of soil-borne diseases of corn is remarkable, with an average of more than 60%.

Compared with the prior art, the invention has the advantages that: 1. the 7richoderma-based composite bio-seed coating agent not only contains 7richoderma chlamydospore fermentation broth, but also contains chitosan oligosaccharide and phenazine-1-

carboxylic acid, two biological pesticide components; the results showed that Trichoderma,5p2648 fermentation broth could inhibit Fusarium spp, Rhizoctonia solani and Pythium spp, and induce corn resistance, chitosan oligosaccharide could induce corn resistance, while phenazine-1-carboxylic acid could inhibit Rhizoctonia solani; 2. Trichoderma-based bio-seed coating agent is a new type of seed coating agent for controlling soil-borne diseases of corn, which is optimized by three biological components and has complementary functional advantages; it not only has obvious control effect on corn stem rot and sheath blight, but also has obvious promotion effect on plant growth index, improving plant stress resistance and increasing crop yield; 3. according to the 7richoderma-based biological seed coating agent provided by the invention, corn seeds are coated and sown according to a certain proportion, and there is no pollution to the soil; field trials in Nanchong, Sichuan, Yinchuan, Ningxia, Shijiazhuang, Guizhou, Zunyi,

Dandong, Liaoning, Nanjing, Jiangsu and other places showed that the control effect of

Trichoderma biological seed coating agent on stalk rot and sheath blight of corn could reach more than 60%, and the yield of corn increased by more than 10%; 4. the invention is suitable for most 7richoderma strains with good biocontrol effect, with simple manufacturing method, low cost, suitable for different scale production and broad market prospect.

BRIEF DESCRIPTION OF THE FIGURES

Other features, objects and advantages of the invention will become more apparent from a reading of the detailed description of non-limiting embodiments made with reference to the following accompanying drawings.

Fig. 1 is a schematic diagram of the results of the screening of film-forming agent types

Fig. 2 is a schematic diagram of the results of the shelf-life determination of the seed coating agents of the present invention.

DESCRIPTION OF THE INVENTION

The present invention will be described in detail with reference to the following specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but will not limit the present invention in any way. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These all belong to the scope of protection of the present invention.

Embodiment 1

1. Plate strain activation: Trichoderma harzianum strainSH2303 is inoculated into PDA plate, 500648 and cultured upside down in an incubator at 28°C for 4-5 days.

PDA culture medium: 200 g potato juice filtered after cooking, 20 g glucose, 20 g agar powder, purified water added to the final total volume of 1 L, packed into 500 mL triangular bottle every 250 mL, autoclaved at 121°C for 25 min. 2. Preparation of strain seed liquid: 200 g potato, 20 g glucose, 1 L purified water, each 250 mL is packed into a 500 mL triangle bottle, and autoclaved at 121°C for 25 min; inoculating culture discs taken from plate culture, and culturing in a shaker at 180 rpm and 28°C for 5 days to obtain

Trichoderma harzianum strain SH2303 liquid fermentation cultures. 3. Trichoderma strain liquid fermentation: 8 kg of corn flour, 80 g of magnesium sulfate, 1.2 g of ferrous sulfate, 0.4 g of manganese sulfate, 0.32 g of zinc sulfate, 0.61 kg of potassium dihydrogen sulfate, 0.23 kg of sodium nitrate, 176 g of ammonium nitrate, 160 g of calcium chloride, 200 L of water, and put into a 300 L fermentor. The initial pH of the culture medium is adjusted to 4-5, sterilized at 21°C for 40-60 min. The final Trichoderma strain chlamydospore fermentation broth is obtained by inoculating 0.5% (v/v)Trichoderma strain seed liquid at 28°C for 7 days. 4. Preparation of Trichoderma-based biological seed coating agent: 1 L of Trichoderma fermentation broth, 4 g of chitosan, 1 mg of phenazine-1-carboxylic acid, and 0.6 g of humic acid, fully mixed. The above mixture is mixed with 13% (W/V) film-forming agent, which is a

Trichoderma-based biological seed coating agent . 5. Trichoderma-based biological seed coating agent can promote the growth of maize seedlings.

The results of pot experiment in greenhouse showed that compared with the control, the seed emergence rate, plant height and taproot length after seed coating agent treatment increased to a certain extent, with the increase rates of 6.32%, 9.84% and 3.34% respectively, but there was no significant difference between them (P > 0.05). Other growth indexes were significantly different from the control (Table 3).

Table 3 Effects of bio-seed coating agent on corn growth

Embodiment 2 LU502648 1. Plate strain activation: Trichoderma harzianum SH2303 is inoculated into PDA culture plate, and cultured upside down in an incubator at 28°C for 4-5 days.

PDA culture medium: 200 g potato juice filtered after cooking, 20 g glucose, 20 g agar powder, purified water added to the final total volume of 1 L, packed into 500 mL triangular bottle every 250 mL, autoclaved at 121°C for 25 min. 2. Preparation of the strain seed liquid: 200 g potato, 20 g glucose, 1 L purified water, each 250 mL is packed into a 500 mL triangle bottle, and autoclaved at 121°C for 25 min; inoculating culture discs taken from plate culture, and culturing in a shaker at 180 rpm and 28°C for 5 days to obtain Trichoderma seed strain liquid culture. 3. Trichoderma strain liquid fermentation: 8 kg of corn flour, 80 g of magnesium sulfate, 1.2 g of ferrous sulfate, 0.4 g of manganese sulfate, 0.32 g of zinc sulfate, 0.61 kg of potassium dihydrogen sulfate, 0.23 kg of sodium nitrate, 176 g of ammonium nitrate, 160 g of calcium chloride, 200 L of water, and put into a 300 L fermentor. The initial pH of the culture medium was adjusted to 4-5; sterilized at 21°C for 40-60 min. The final 7richoderma strain chlamydospore fermentation broth is obtained by inoculating 0.5% (v/v)Trichoderma strain seed liquid at 28°C for 7 days. 4. Preparation of Trichoderma-based bio-seed coating agent: 1 L of Trichoderma strain fermentation broth, 4 g of chitosan, 1 mg of phenazine-1-carboxylic acid and 0.6 g of humic acid, fully mixed. The above mixture is mixed with 17%(W/V)853 film-forming agent, which is the

Trichoderma-based biological seed coating agent, seed dressing according to the ratio of seed coating agent to seed of 1:10 (W/W); the blank seed coating agent (only film-forming agent) is used as the control. 5.Field plot test of biological seed coating agent: Fusarium spp or Rhizoctonia solani is cultured in wheat grain culture medium for 10 days as pathogen inoculum, respectively. When corn is sown, 2 g of pathogen inoculum is inoculated around the seeds, and 50 holes are inoculated in each treatment, which is repeated for three times. The blank control without any seed coating agent is used as the control. When corn enters the filling stage, the incidence of corn stalk rot is investigated and the control effect is calculated.

Incidence rate% = number of diseased plants/total number of investigated plants x 100

Control effect% = [(incidence of control field - incidence of treated field)/incidence of control field] x 100

The field plot test show that the disease index of biological seed coating agent decreased from 70 to 45.83; the number of wilt leaves and early aging rate of leaf were lower than those of the control, and the difference is extremely significant (P < 0.01). Plant lodging rate is lower than,5p2648 that of the control, and the difference is significant (p < 0.05). The ear drooping rate is also lower than that of the control, but the difference is not significant (p>0.05). The results show that the seed coating agent could reduce the occurrence of corn stalk rot (Table 4). Further investigation shows that 7richoderma-based biological seed coating agent has obvious control effect on stalk rot and sheath blight, with 57.35% control effect on sheath blight and 75% control effect on stem rot.

Table 4 Control effect of bio-seed coating agent on corn stalk rot leaves (pieces leaves (pieces rate (% rate (% rate (%

Table 5 Control effect of bio-seed coating agent on sheath blight and stem rot rate (% % % %

Toc ww [= sw =

Embodiment 3 1. Strain growth activated in plate: inoculate Trichoderma harzianum strain SH2303 into PDA culture dish, and culture it upside down at 28°C for 4-5 days in an incubator.

PDA culture medium: 200 g potato juice filtered after cooking, 20 g glucose, 20 g agar powder, purified water added to final total volume of 1 L, subpackaged into 250 mL triangular bottle, autoclaved at 121°C for 25 min. 2. Preparation of the strain seed liquid: 200 g potato, 20 g glucose, 1 L purified water, subpackaging into a 250 mL triangular flask, autoclaving at 121°C for 25 min; inoculating strain culture discs taken from plate culture, and culturing in a shaker at 180 rpm and 28°C for 5 days to obtain Trichoderma harzianum strain SH2303 liquid fermentation strain. 3. Trichoderma strain liquid fermentation: 8 kg of corn flour, 80 g of magnesium sulfate, 1.2 g of ferrous sulfate, 0.4 g of manganese sulfate, 0.32 g of zinc sulfate, 0.61 kg of potassium dihydrogen sulfate, 0.23 kg of sodium nitrate, 176 g of ammonium nitrate, 160 g of calcium chloride, 200 L of water, and put into a 300 L fermentor. The initial pH of the culture medium is adjusted to 4-5; sterilized at 21°C for 40-60 min. The final Trichoderma chlamydospore fermentation broth is obtained by inoculating 0.5% (v/v) Trichoderma strain seed liquid at 28°C for 7 days.

4. Preparation of Trichoderma-based biological seed coating agent: 1 L of Trichoderma strain,502648 fermentation broth, 4 g of chitosan, 1 mg of phenazine-1-carboxylic acid, and 0.6 g of humic acid, fully mixed. The above mixture is mixed with 14% (W/V) 853 film-forming agent, which is the 7richoderma-based biological seed coating agent; seed dressing according to the ratio of seed coating agent to seed of 1: 15 (W/W). 5. Field application trials: the test areas are arranged in Zunyi, Guizhou, Nanchong, Sichuan,

Shijiazhuang, Hebei and other places. The locally grown corn varieties are selected with uniform in size and normal germination rate; trial plots with serious diseases and insect pests occurred in previous years, flat terrain, not prone to farmland ponding and early drought and flood; The experimental design adopts random block arrangement. The results show that Trichoderma biological seed coating agent has good control effect on these three diseases (Table 6).

Table 6 Effect of 7richoderma-based bio-seed coating agent on controlling soil-borne diseases

The component of that seed coating agent of the invention have different degrees of inhibition and antagonism to different pathogens. The comparative study of different concentration ratios in this experiment show that chitosan concentration of 3.5-4.5 mg/mL and phenazine-1-carboxylic acid concentration of 1 ug /mL had obvious inhibitory effect on Fusarium, and the inhibitory rate is 53.3%. Following this concentration ratio, together with 7richoderma strain chlamydospore fermentation broth, has an inhibition effect of 62.9% on Fusarium graminearum. On this basis, the inhibitory effect of humic acid with different proportions on pathogens is obviously different.

When the humic acid content is in the range of 0.05-0.1% (W/V), the control effect of the combination ratio on pathogen is extremely significant compared with the control, and the control effect is also significantly different from that of the ratio without humic acid, and the control effect is obviously improved, reaching 73.5%. However, when the concentration of humic acid is lower than 0.05% (W/V) or higher than 0.1% (W/V), the control effect decreases obviously, and even the addition of humic acid has some adverse effects on other component combination ratio against pathogen inhibition . Therefore, the 7richoderma-based seed coating agent prepared by strictly adding humic aids 0.05-0.1% (W/V) into the ratio has a very obvious control effect on pathogenic bacteria. (Tables 7-1 and 7-2)

Table 7-1 Effects of different concentrations of compound bio-seed coating agent on Fusarium 502648 growth ; ; Diameter

Different compound concentrations (mm)

Treatments 7 7 ——

Chitosan phenazine-1-carboxylic acid 96h (mg/mL) (ug/mL) 30.6Aa . B | 2403 | dl | 263Ab 8Ba ; D | 40s |__| | 18.5Bb 20.2Ba 19.3Ba . CK | 0 |. 0 | 396Aa

Trichodermaagent | 0 |. 0 [| 18.8Bb agent

Table 7-2 Effects of different concentrations of compound bio-seed coating agent on Fusarium growth

Humic acid (g/L)

LD | 180Bb 0.2 Trichoderma agent 19.3Ba

D+ Trichoderma agent 18.5Ba 0 | 305Aa

LD | 175Bb

Trichoderma agent 15.9Bc

D+ Trichoderma agent 10.5Cb

L.. 0 | 199Ba ben 12 Trichoderma agent 20.0Ba

D+ Trichoderma agent 17.3Bb 0 | 255Ab oo | 0 | 396Aa

At the same time, the components of the seed coating agent of the invention can promote the growth of corn to a certain extent. Chitosan concentration of 3.5-45 mg/mL and phenazine-1-carboxylic acid concentration of 1 ug /mL can promote the growth of maize radicle and embryo, but the promotion effect is not obvious. However, this concentration ratio if further combined with Trichoderma strain chlamydospore fermentation broth can obviously promote the growth of corn germ and radicle. The growth of germ is twice that of the control, and the radicle elongation is 35.2%. After adding a certain proportion of humic acid in the above combined ratio, the synergistic effect is extremely remarkable, with the elongation of radicle increasing by 52.6% and the elongation of embryo increasing by 42.3%. However, the addition of humic acid beyond

0.05-0.1% (W/V) has no obvious effect on promoting the growth of corn radicle and germ, anq;502648 even has a tendency to inhibit the growth. This shows that humic acid has synergistic effect with

Trichoderma in the plant growth-promotion at a certain range. (Tables 8-1 and 8-2)

Table 8-1 Effects of different component ratios on the growth of maize radicle and germ

Different compound concentrations

Chitosan phenazine-1-carboxylic acid Germ Radicle

Treatments (mg/mL) (ug/mL) (cm) (cm)

BB | 2403 | dl 36 [| 64 bo | 40s | dl 42 [| 81

EB | 605 | 01 23 | 68 . CK | 0 0 27 [ 71

Trichoderma agent | 0 | 0 A2 | 8

Trichoderma.agent +D 4+0.5 1 5.7

Table 8-2 Effects of different components ratios on the growth of maize radicle and germ bo | 31 | 73 1 02

D+ Trichoderma agent oo | 25 | 64

LD | 43 | 08

Trichoderm agent

D+ Trichoderma agent oo 39 | 91

LD | 3 [| 72 12 Trichoderma agent |__ 29 | 68

D+ Irichodermaagent. | 3 | 69 oo | 25 | 1 oo | 0 | 27 | 71

The bio-seed coating agent is prepared by optimizing the ternary combination formula and mixing it with carriers such as film forming agent. The preparation method is simple and low in cost, and it is suitable for different scale production.

According to the invention, after optimizing the combined formula, the bio-seed coating agent is prepared by mixing with a film forming agent according to a certain proportion. Through experiments, different film-forming agents and their contents have certain influence on the disease prevention effect of seed coating agents. After screening, a film-forming agent which has no influence on the growth of biocontrol microbe Trichoderma strain is selected (Fig. 1). The experiment shows that the film-forming agent 853 has no adverse effect on the growth pf)502648

Trichoderma strain and even has a certain promoting effect.

According to the invention, the optimized formula is mixed with 853 film-forming agents with different concentrations, and the bacteriostatic rate of Fusarium spp and Rhizoctonia solani is measured (Table 9). The results showed that 853 film-forming agent with different concentrations had obvious effects on the inhibition of pathogens after being mixed with the above optimized formula. When the concentration of film-forming agent is in the range of 13-17%(W/V), the antagonistic rate is 68%, with a significant difference. However, when the concentration range is lower or higher than this range, there is no significant difference or even no significant difference between the inhibition radius and the control. This indicates that a certain concentration of film-forming agent (acrylamide modified synthetic polymer) may have synergistic effect with the above-mentioned optimized ingredients by promoting the production of certain unknown substance or antagonistic effect of 7richoderma ,which thereby significantly increasing the inhibition rate of pathogens .

Table 9 Effects of different concentrations of film-forming agents and optimized components on pathogens

EE

The seed coating agent of the invention is mixed with corn seeds according to different proportions, and the effects of different mixing treatments on the control effect of soil-borne diseases in corn seedling stage are observed, so as to determine the seed coating agent-seed ratio.

As can be seen from the following table, there are differences among coating treatments with different drug ratios. The coating treatment with the ratio of 1:10 has the highest control effect against stalk rot and sheath blight in maize seedling stage, with the relative control effects of 78% and 67.59% respectively. There is a big difference in the control effect between different dosage treatments of Trichoderma-based coating alone, and the relative control effect is the highest when the ratio of seed coating agent:seed is 1:20, and the correlation between control effect and dosage is not significant. Results ( e.g. (1) in Table 10) showed that the disease index of sheath blight and stalk rot in the treatment of seed coating agent : seed ratio of 1:10 is much lowest which are 17.50 and 16.00, respectively, and the relative control effect is the highest, 67.59Ÿ4,502648 and 78.00%, respectively. Comprehensive analysis shows that 7richoderma-based seed coating agent can reduce the disease index of maize at seedling stage, and the optimal ratio of seed coating agent and seed is 1:10, which also lays a good foundation for improving corn growth and disease resistance in the late corn growth stages(Table10).

Table 10 Control effect of different treatments on soil-borne diseases in corn seedling stage

SL index % index %

Co [sw | ww

Note: treatments (1)-(3) are seed coating agents used at the ratio of 1:10, 1:20 and 1:40, respectively; (4)-(6) coating seeds with Trichoderma solid fermentation biomass according to the ratio of biomass to seed of 1:10, 1:20 and 1:40; (7) chemical pesticide, carbendazim; blank control (CK). “-” for not present.

Embodiment 4 1. Plate strain activation: inoculate Trichoderma harzianum strain SH2303 into PDA culture plate, and culture it upside down at 28°C for 4-5 days in an incubator.

PDA culture medium: 200 g potato juice filtered after cooking, 20 g glucose, 20 g agar powder, purified water added to the final volume of 1 L, subpackaged into 250 mL triangular bottle, autoclaved at 121°C for 25 min. 2. Preparation of the strain seed liquid: 200 g potato, 20 g glucose, 1 L purified water, subpackaging into a 250 mL triangular flask, autoclaving at 121°C for 25 min; inoculating the plate strain, and culturing in a shaker at 180 rpm and 28°C for 5 days to obtain Trichoderma harzianum strain SH2303 liquid fermentation cultures. 3. Trichoderma liquid fermentation broth: 8 kg of corn flour, 80 g of magnesium sulfate, 1.2 g of ferrous sulfate, 0.4 g of manganese sulfate, 0.32 g of zinc sulfate, 0.61 kg of potassium dihydrogen sulfate, 0.23 kg of sodium nitrate, 176 ammonium nitrate, 160 g of calcium chloride, 200 L of water, and put into a 300 L fermentor. The initial pH of the culture medium was adjusted to 4-5; sterilize at 21°C for 40-60 min. The final Trichoderma strain chlamydospore;5p2648 fermentation broth is obtained by inoculating 0.5% (v/v)Trichoderma strain seed liquid at 28°C for 7 days. 4. Preparation of Trichoderma-based seed coating agent: 1 L of Trichoderma strain fermentation broth, 4 g of chitosan, 1 mg of phenazine-1-carboxylic acid, and 0.6 g of humic acid, fully mixed.

The above mixture is mixed with 14% (W/V) film-forming agent, which is 7richoderma-based bio-seed coating agent. 5. Determine the shelf life of the above bio-seed coating agent. The initial spore content of

Trichoderma harzianum chlamydospore fermentation broth is 2.5x10® CFU/mL, and chitosan is not added as seed coating agent. The results show that chitosan in seed coating agent can not only inhibit pathogens growth, but also provide sustained nutrition for 7richoderma growth and sporulation, which can also prolong the seed coating shelf life. After 6 months, the spore amount in seed coating agent can still reach 1.56x10$ CFU/g, as shown in Fig. 2.

The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which will not affect the essential content of the present invention.

Claims (10)

CLAIMS LU502648

1. A Trichoderma-based seed coating agent, characterized in that the seed coating agent comprises Trichoderma chlamydospore fermentation broth, film forming agent, chitosan oligosaccharide, phenazine-1-carboxylic acid and humic acid; wherein, each liter of Trichoderma chlamydospore fermentation broth contains 13-17% of film-forming agent, 0.35-0.45% of chitosan oligosaccharide, 0.5-1.5 ug/mL of phenazine-1-carboxylic acid and 0.05-0.1% of humic acid, and the percentages are mass-volume ratio.

2. The Trichoderma-based seed coating agent according to claim 1, characterized in that the film-forming agent comprises 853 film-forming agent.

3. The Trichoderma-based seed coating agent according to claim 1, characterized in that the Trichoderma is Trichoderma harzianum strain SH2303.

4. The Trichoderma-based seed coating agent according to claim 1, characterized in that the preparation of the Trichoderma chlamydospore fermentation broth comprises the following steps: inoculating the first-class strain into the 7richoderma liquid fermentation medium with an inoculation amount of 0.5% strain seed liquid , and fermenting or culturing at 28°C for 7 days; the strain seed liquid is obtained by culturing Trichoderma discs in PD culture medium at 180 rpm and 28°C for 5 days with the inoculation amount of 3-5 discs pieces/triangular flask.

5. The Trichoderma-based seed coating agent according to claim 4, characterized in that the composition of the Trichoderma liquid fermentation medium comprises 40 kg of corn flour, 400 g of magnesium sulfate, 6.0 g of ferrous sulfate, 2.0 g of manganese sulfate, 1.6 g of zinc sulfate,

3.06 kg of potassium dihydrogen sulfate, 1.13 kg of sodium nitrate, 880 g of ammonium nitrate and 800 g of calcium chloride per 1,000 L of Trichoderma liquid fermentation medium, the balance is water and the initial pH is adjusted to 4-5.

6. The Trichoderma-based seed coating agent according to claim 4, characterized in that the tank loading amount of the fermentation culture is controlled in that 200 L liquid fermentation medium is loaded into a 300 L fermentor.

7. A preparation method of the seed coating agent according to any one of claims 1 to 6, characterized by comprising the following steps: mixing chitosan, phenazine-1-carboxylic acid, humic acid and Trichoderma chlamydospore fermentation broth according to the proportion, and then adding a film-forming agent to mix to obtain the seed coating agent.

8. Application of the seed coating agent according to any one of claims 1 to 6 as a biocontrol agent in controlling soil-borne diseases of corn.

9. The application according to claim 8, characterized in that the soil-borne diseases include,5p2648 controlling corn stalk rot, sheath blight and root rot.

10. A method for using the Trichoderma-based seed coating agent in controlling soil-borne diseases of corn according to any one of claims 1 to 6, characterized in that the Trichoderma-based seed coating agent and corn seeds are uniformly mixed according to the mass ratio of 1: (10-15).