Pesticide composition containing trichoderma harzianum and purpureocillium lilacinum and application thereof

Abstract

The invention relates to a pesticide composition containing trichoderma harzianum and purpureocillium lilacinum and application of the pesticide composition, and belongs to the field of biological pesticides. The pesticide composition is granules containing trichoderma harzianum and purpureocillium lilacinum, and specifically comprises 2% of trichoderma harzianum YB01 pure sporopollen, 1% of purpureocillium lilacinum PlGZ490 pure sporopollen, 10% of an adhesive, 2% of a disintegrating agent and 85% of a carrier. The granule provided by the invention can significantly reduce the occurrence of cucumber fusarium wilt and tomato root knot nematode disease, can promote the growth of crops, can reduce the use of chemical pesticides, is safe to the environment, and has good economic, ecological and social benefits.

Description

Technical Field

[0001] The invention relates to a pesticide composition containing Trichoderma harzianum and Pseudomonas lilacinus and application thereof, belonging to the field of biological pesticides. Background Art

[0002] Soil is the matrix on which plants grow and is also a necessary resource for human survival and development. Farmland degradation and continuous cropping problems seriously affect crop production. The abnormal distribution of soil microorganisms and the outbreak of soil-borne plant diseases and insect pests are the causes or manifestations of farmland degradation and continuous cropping problems. Appropriate supplementation of soil microorganisms is becoming an effective way to ensure healthy crop production.

[0003] Fusarium wilt is a common soil-borne disease that seriously harms crop production. It is common in many crops around the world, with melons, cotton, bananas and other crops being the worst affected. Plant root-knot nematode disease is also an important soil-borne disease. Root-knot nematodes are highly specialized omnivorous plant pathogenic nematodes that feed on rhizomes in the soil, destroying the normal tissue structure of the plant roots. Root-knot nematode disease and wilt are closely related. Nematodes cause many wounds on the roots of plants, creating favorable conditions for the infection of wilt, thereby aggravating the incidence of the disease. In order to prevent and control the above two soil-borne diseases, chemical pesticides have to be used in large quantities, which causes environmental pollution and excessive pesticide residues in food. Therefore, microbial pesticides are also being developed and applied. A search of my country's pesticide registration information shows that fungal products used to control Fusarium wilt include Trichoderma sp., bacteria include Bacillus subtilis, Bacillus polymyxa, Bacillus licheniformis, Bacillus amyloliquefaciens, etc.; fungal pesticide products used to control root-knot nematodes include Paecilomyces lilacinum (now known as Purpureocillium lilacinum) and Verticillium chlamysporum, and bacterial products include Bacillus firmus, Rhodomyces thiophilus HNI-1, Methylotrophic Bacillus LW-6, Bacillus cereus, Bacillus nematicidal B16 and Bacillus thuringiensis HAN055, etc. At present, there is no registered microbial pesticide in my country that can simultaneously control Fusarium wilt and root-knot nematodes.

[0004] There are more than 40 related invention patents on microorganisms for preventing and controlling wilt diseases, but most of them use bacteria to prevent and control wilt diseases, such as CN202311133668.9, CN202210555584.3, CN202110259568.5, CN201911150894.1, CN202011240300.9 and CN201811098315.9, etc., which disclose the application technology of various Bacillus for preventing and controlling various wilt diseases. Patents such as CN201810830048.3, CN201910552381.7, CN202211153419.1, and CN202010772722.4 disclose the prevention and control technology and application of various actinomycetes for wilt diseases. Currently, there are four patents disclosed for using fungi to control wilt: CN201711194381.1 discloses the As-75 strain of Aspergillus sclerotiorum, its fermentation culture solution and application; CN201610172486.6 discloses a composite bacterial agent of green fleshy sophora for controlling cucumber wilt and its preparation method; CN202311753128.0 discloses a new strain TB2 of Trichoderma brevis and its application; and CN201310144073.3 discloses a strain of Trichoderma acanthosporum and its application. There are also many patent applications for microbial control technology of root-knot nematodes, but the vast majority of the published patents are related to bacteria and actinomycetes, such as CN202310951417.5 discloses a Streptomyces fossa, a Streptomyces fossa oil suspension and its preparation method and application, CN202210798198.7 discloses a strain of Brevundimonas BWT19-19 and its application in controlling plant root-knot nematodes, and CN202210253588.6 discloses a strain of Bacillus subtilis 2JQ3 with toxic activity against root-knot nematodes and its application, etc.

[0005] Further search and analysis of various literature and patent databases revealed that current research progress mainly focuses on using different microorganisms to control Fusarium wilt or root-knot nematode disease alone, and microbial technology that can simultaneously control Fusarium wilt and root-knot nematodes has not been reported. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a pesticide composition containing Trichoderma harzianum and Pseudomonas lilacinus and application thereof.

[0007] The purpose of the present invention is to develop a microbial pesticide composition mixed with two biocontrol fungi to achieve the purpose of simultaneously preventing and controlling wilt disease and root-knot nematode disease, so as to reduce the use of chemical pesticides, improve crop quality and environmental safety, and enhance the economic, ecological and social benefits of agricultural production.

[0008] To achieve the above-mentioned purpose, the technical scheme adopted by the present invention is as follows: on the one hand, the present invention provides a pesticide composition containing Trichoderma harzianum and Pseudomonas lilacinus, the active ingredients of the granules include pure spore powder of Trichoderma harzianum YB01 and pure spore powder of Pseudomonas lilacinus PlGZ4903; the sum of the masses of the pure spore powder of Trichoderma harzianum YB01 and the pure spore powder of Pseudomonas lilacinus PlGZ4903 is 3% of the total mass of the pesticide composition; the mass ratio of the pure spore powder of Trichoderma harzianum YB01 to the pure spore powder of Pseudomonas lilacinus PlGZ4903 is (1-3):(1-3).

[0009] In some embodiments of the present invention, in the pesticide composition, the total amount of pure spore powder added is 3% by percentage. The mass ratio of the pure spore powder of Trichoderma harzianum YB01 to the pure spore powder of Pseudomonas lilacinus PlGZ4903 is 1:1, 2:1, 3:1, 1:2, 1:3. Preferably, the mass ratio of the pure spore powder of Trichoderma harzianum YB01 to the pure spore powder of Pseudomonas lilacinus PlGZ4903 is 2:1.

[0010] In another aspect, the present invention provides a granule, which comprises the above-mentioned pesticide composition and an adjuvant; the adjuvant comprises a carrier, a binder and a disintegrant.

[0011] In some embodiments of the present invention, the granules include the following components, measured in percentage: 0.75-2.25% pure spore powder of Trichoderma harzianum YB01, 0.75-2.25% pure spore powder of Pseudomonas lilacinus PlGZ490, 10% binder, 1-3% disintegrant and the remainder of carrier.

[0012] In some embodiments of the present invention, the granules include the following components, measured in percentage: 2% pure spore powder of Trichoderma harzianum YB01, 1% pure spore powder of Pseudomonas lilacinus PlGZ490, 10% binder, 2% disintegrant and 85% carrier.

[0013] In some embodiments of the present invention, the carrier includes at least one of wheat bran, bamboo powder, kaolin, diatomaceous earth, and white carbon black, the binder includes dextrin or gelatin, and the disintegrant is xanthan gum.

[0014] In some embodiments of the present invention, the carrier is wheat bran and the binder is dextrin.

[0015] In another aspect, the present invention provides use of the granules in preventing or controlling crop wilt or root-knot nematode disease.

[0016] In some embodiments of the present invention, the crop wilt disease is cucumber wilt.

[0017] In some embodiments of the present invention, the root knot nematode disease is tomato root knot nematode disease.

[0018] Compared with the prior art, the present invention has the following beneficial effects: the fungal granules provided by the present invention are compounded by two biocontrol fungi (Trichoderma harzianum YB01 and Pseudomonas lilacinus PlGZ4903) in a specific mass ratio of 2:1, and can simultaneously prevent and control wilt disease and root-knot nematode disease, thereby improving efficacy, increasing crop yields and significantly reducing the use of chemical pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a safety test of Trichoderma harzianum and several strains of Pseudomonas lilacinus on plants, among which, Figure 1 a is the lilacinus strain PlQS01; b is the lilacinus strain PlYN8501; c is the lilacinus strain PlGX17A01; d is the lilacinus strain PlGZ4203; e is the lilacinus strain PlB03B01; f is the lilacinus strain PlGX1702; g is the lilacinus strain PlGX06A01; h is the lilacinus strain PlGX0401; i is the lilacinus strain PlSC59A3; j is the lilacinus strain PlGZ4903; k is the lilacinus strain PlHN17; l is Trichoderma harzianum YB01; and m is CK.

[0020] Figure 2 It is a confrontation culture of Pseudomonas lilacinus and Trichoderma harzianum. The central bacteria of all plates are Trichoderma harzianum, and the surrounding bacteria are Pseudomonas lilacinus. Figure 2 In the figure, a is the lilacinus strain P1SC61B03; b is the lilacinus strain P1B20A01; c is the lilacinus strain P1GX77A04; d is the lilacinus strain P1GZ4203; e is the lilacinus strain P1QS01; f is the lilacinus strain P1GX0401; g is the lilacinus strain P1GX7706; h is the lilacinus strain P1GX17A01; i is the lilacinus strain P1GX2801; j is the lilacinus strain P1GX1702; k is the lilacinus strain P1B03B01; l is the lilacinus strain P1YN8501; m is the lilacinus strain P1HN17; n is the lilacinus strain P1GZ4903; o is the lilacinus strain P1SC59A3; p is the lilacinus strain P1GX06A01; q is CK.

[0021] Figure 3 The culture medium is a confrontation culture of Trichoderma harzianum and Pseudomonas lilacinus PlGZ4903 and PlSC61B03, wherein: Figure 3a1 shows that the growth process of the purpurogenous spore strain P1GZ49033 has an inhibition zone; a2 shows that the purpurogenous spore strain P1GZ4903 produces spores in the later stage; b1 shows that the growth process of the purpurogenous spore strain P1SC61B03 has no inhibition zone; b2 shows that the purpurogenous spore strain P1SC61B03 does not produce spores in the later stage.

[0022] Figure 4 The antibacterial effect of Trichoderma harzianum YB01 strain on pathogenic bacteria, among which, Figure 4 a and b in the figure are the confrontation experiment of Fusarium solani; c and d are the confrontation experiment of Fusarium oxysporum; e and f are the confrontation experiment of cucumber wilt pathogen. The first row (a / c / e) is the control group pathogen, and the second row (b / d / f) is the treatment group (pathogen in the center and Trichoderma harzianum in the four corners).

[0023] Figure 5 The biocontrol effects of different P. lilacinus strains on root-knot nematodes are shown in Table 1. Figure 5 a is the lilacinus strain PlQS01; b is the lilacinus strain PlYN8501; c is the lilacinus strain PlGX17A01; d is the lilacinus strain PlB03B01; e is the lilacinus strain PlGX1702; f is the lilacinus strain PlGX06A01; g is the lilacinus strain PlSC59A3; h is the lilacinus strain PlGZ4903; i is the lilacinus strain PlHN17; and j is CK.

[0024] Figure 6 The effect of Pseudomonas lilacinus PlGZ4903 on tomato roots, among which, Figure 6 a in the figure is CK; b is Pseudomonas lilacinus PlGZ4903.

[0025] Figure 7 It is a finished product of 3% Trichoderma harzianum and Pseudomonas lilacinus granules.

[0026] Figure 8 These are colonies formed on a PDA plate by the finished product of 3% Trichoderma harzianum·Purplesporium lilacinum granules.

[0027] Fig. 9 The growth of cucumber after applying different formulations of Trichoderma harzianum and Pseudomonas lilacinus granules for 7 days. Fig. 9 a is a clear water control; b is the granules of comparative example 1; c is the granules of comparative example 2; d is the granules of comparative example 3; e is the granules of comparative example 4; f is the granules of example 3; g is the granules of example 5; h is the granules of example 6; i is the granules of example 7; and j is the granules of example 8. DETAILED DESCRIPTION

[0028] In order to better illustrate the purpose, technical scheme and advantages of the present invention, the present invention will be further described below in conjunction with specific drawings and embodiments. In the embodiments, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0029] Example 1: This example is the screening of active ingredients of the pesticide composition of the present invention

[0030] 1. Safety determination of Trichoderma harzianum and Pseudomonas lilacinus on plants

[0031] The Trichoderma harzianum and Pseudomonas lilacinus blocks were inoculated into PDB medium and cultured in a shaking incubator at 26°C and 180 r / min for 3 days. The spore suspension was enriched by centrifugation after filtering with gauze and diluted with 0.02% Tween-80 to a spore concentration of 10 7 spores / mL for later use. Use a sterilized pipette tip to drill 5 small holes with a diameter of 1 cm evenly within 2 cm around the base of the cucumber stem. Drip 6 mL of spore suspension into each hole. A total of 30 mL of spore suspension was irrigated into each pot. Six replicates were made for each treatment. 0.02% Tween-80 was used as the control. The growth of the cucumber seedlings was observed after 7 days. The results are shown in Figure 1 .

[0032] Experimental results: From Figure 1 It can be seen that the cucumber seedlings in each treatment group grew healthily, and the cucumber leaves showed no signs of yellowing, wilting or drying up, indicating that Trichoderma harzianum and multiple strains of Pseudomonas lilacinus had no negative impact on the plants and could be used to make granular products.

[0033] 2. Compatibility determination of Trichoderma harzianum and Pseudomonas lilacinus

[0034] The plate confrontation method was used for the test: a 5mm punch was used to punch out a cake of Trichoderma harzianum, inoculated in the center of the PDA plate, a block of Psoralea lilacinus was taken and inoculated 3 cm away from the block of Trichoderma harzianum, and a control was used without the cake of Psoralea lilacinus. The plate was cultured in a 26±1℃ incubator for 1-2 days until the block of Trichoderma harzianum filled the culture dish, the colony diameter of Trichoderma harzianum and the colony diameter of the pathogen in the control group were measured, and the average inhibition rate was calculated according to the formula after recording the data. Inhibition rate (%) = [(diameter of the control group - diameter of the treatment group) / (diameter of the control group - diameter of the cake)] × 100. Each treatment was repeated three times, and the data were analyzed using SPSS16 software. The results are shown in Table 1. Figure 2 and Figure 3 .

[0035] Table 1 The inhibition rate of Pseudomonas lilacinus to Trichoderma harzianum

[0036]

[0037] Note: The data in the table are mean ± standard deviation. Different letters after the data in the same column indicate significant differences at the P < 0.05 level (DMRT method).

[0038] Experimental results: In the confrontation culture experiment, it was found that an inhibition zone would form between some of the purpurogenous fungi and Trichoderma harzianum. The purpurogenous fungi with an inhibition zone would grow normally and produce spores in the later stage, while the purpurogenous fungi without an inhibition zone would not produce spores in the later stage. Figure 3 As shown in . Therefore, in order to ensure the normal growth and reproduction of Pseudomonas lilacinus in the mixed granules, the strains PlSC61B03, PlB20A01, PlGX77A04, PlGX7706, and PlGX2801 without inhibition zones were selected and the other strains with inhibition zones were retained for subsequent experiments. It can also be seen from the experimental results in Table 3 that Pseudomonas lilacinus PlGZ4203 and PlGX0401 have a greater effect on the growth of Trichoderma harzianum. The difference in colony diameter after confrontation is significant, and the average inhibition rates can reach 49.16% and 32.51%, respectively. The remaining strains have little effect on the growth of Trichoderma harzianum. The colony diameter after confrontation is no different from that of CK, and the inhibition rates are all lower than 25%. The confrontation experiment pictures of the remaining strains are shown in Figure 2 shown.

[0039] 3. Determination of the inhibitory effect of Trichoderma harzianum on plant pathogenic fungi

[0040] The plate confrontation method was used for the test: Fusarium solani, Fusarium oxysporum, and Fusarium oxysporum f.sp.cucumerinum were taken with a 5mm puncher and inoculated in the center of the PDA plate. Four Trichoderma harzianum YB01 strain blocks were inoculated in a square at 3 cm around the pathogen blocks. The control plate was not inoculated with Trichoderma harzianum and placed in a 26°C incubator for 1 to 2 days until the pathogen colonies in the control group filled the culture dish. The diameter of the pathogen colonies and the diameter of the pathogen colonies in the control group were recorded, and the colony diameter = the sum of the vertical and horizontal lengths ÷ 2. The inhibition rate was calculated based on the obtained diameter, and the inhibition rate (%) = [(control group diameter - treatment group diameter) / (control group diameter - bacterial cake diameter)] × 100, and each treatment was repeated 3 times. SPSS 16 software was used to analyze the data, and the results are shown in Table 2 and Figure 4 .

[0041] Table 2 Antibacterial activity of Trichoderma harzianum YB01 strain against plant pathogenic fungi

[0042]

[0043] The data in the table are mean ± standard deviation.

[0044] Experimental results: From the results in Table 2, it can be seen that Trichoderma harzianum YB01 has a good inhibitory effect on three plant pathogenic fungi, namely Fusarium solani, Fusarium oxysporum and Fusarium oxysporum f.sp.cucumerinum. Among them, the inhibitory effect on Fusarium solani is the highest, with an average inhibition rate of 84.75%; the average inhibition rate on Fusarium oxysporum can reach 81.90%, and the average inhibition rate on Fusarium oxysporum can reach 78.65%.

[0045] 4. Biocontrol effect of Pseudomonas lilacinus on root-knot nematodes

[0046] The sterilized and dried soil was mixed with the diseased soil containing root-knot nematodes to prepare a soil containing 15% diseased soil for later use. Tomato seeds were sown in the diseased soil containing root-knot nematodes. When the tomato seedlings grew to two true leaves, 1.0×10 of different strains of Purpureocillium lilacinum were used to inoculate the seedlings. 7 spores / mL suspension was used for treatment. Five small holes with a diameter of 1 cm were evenly drilled within 2 cm around the base of the tomato stem using a sterilized pipette tip. 6 mL of spore suspension was drip-irrigated into each hole. A total of 30 mL of spore suspension was irrigated into each pot of tomato. Clean water was used as a control. Each treatment was repeated 6 times. The growth of tomato plants was observed after 5, 20, and 35 days, and the plant height was measured. SPSS16 software was used to analyze the data. The results are shown in Table 3. Figure 5 as well as Figure 6 .

[0047] Table 3 Biocontrol effects of different P. lilacinus strains on root-knot nematodes

[0048]

[0049] The data in the table are mean ± standard deviation. Different letters after the data in the same column indicate significant differences at the P < 0.05 level (DMRT method).

[0050] Experimental results: From the results in Table 3, it can be seen that the tomato plant height after 35 days of treatment with the PlGZ4903 strain was the highest, reaching 99.83 mm, which was the most obvious difference from the CK group, and was 62.16 mm higher than the average plant height of the CK group. The tomato plant height after 35 days of treatment with the PlGZ4903 strain was also significantly different from that of the other strain treatment groups.

[0051] Depend on Figure 5 It can be seen that the tomato plants in the untreated group CK were short, and all the treatments with P. lilacinus strains had a certain preventive effect on root-knot nematodes, inhibiting the infection of root-knot nematodes and allowing tomatoes to resume growth. Among them, the treatment with strain PlGZ4903 had the best effect. Figure 6It can be seen that the untreated roots of the CK group have yellowed and rotted, and the root system is incomplete, while the roots treated with PlGZ4903 are healthy and complete. 7 spores / mL PlGZ4903 spore suspension has a good control effect on root-knot nematodes. Based on the results in Table 1, it can be seen that PlGZ4903 has the best control effect on root-knot nematodes and has good compatibility with Trichoderma harzianum.

[0052] Based on the exploration and verification of the above experiments, the active ingredient of the pesticide composition of the present invention is finally selected to be a combination of Pseudomonas lilacinus PlGZ4903 and Trichoderma harzianum YB01 strain.

[0053] Example 2: Production and quality detection of pure spore powder of Trichoderma harzianum and Psoralea corylifolia

[0054] Production of pure spore powder of Trichoderma harzianum YB01: Take a small amount of conidia of Trichoderma harzianum YB01 and inoculate it on a PDA plate for activation, culture it in a 26℃ incubator for 3 days, and inoculate the conidia in the plate into the seed culture medium of Trichoderma harzianum. The seed culture medium formula is: 15g sucrose, 15g peptone, 0.5g ammonium sulfate, 1g potassium dihydrogen phosphate, 5g yeast powder, and 1000mL distilled water. Culture it in a shaker at 26℃ and 180r / min for 48h, and inoculate 9% of the seed liquid in the solid matrix culture medium of Trichoderma harzianum for solid fermentation. The solid matrix culture medium is prepared as follows: 375g sucrose, 37.5g peptone, 25g ammonium sulfate, 1.25g potassium dihydrogen phosphate, 12.5g sodium dihydrogen phosphate and 2500mL distilled water are prepared into a nutrient solution, and the nutrient solution is mixed with 1000g coconut bran soil and 100g wheat bran, and then sterilized and bagged for use. Place in a 26°C incubator for 9 days. After the Trichoderma harzianum produces spores, mix it with 0.02% Tween-80 to wash out the spores, filter it with double-layer gauze to obtain a spore suspension, centrifuge it at 5000r / min in a centrifuge for 5 minutes, discard the supernatant, take the precipitate, freeze-dry it for 72 hours, and then package it for later use.

[0055] Production of pure spore powder of P. lilacinus P1GZ4903: A slant is taken to preserve the strain of P. lilacinus P1GZ4903, a small amount of conidia is scraped and inoculated on a PDA plate, and cultured in a 26°C incubator for 7 days, the conidia in the above plate are inoculated into a PDB medium and cultured for 5 days, 13% (w / w) seed liquid is inoculated into sterilized bagged rice for solid fermentation for 7 days, 0.02% Tween-80 is mixed with the rice to wash out the spores, and a spore suspension is obtained by filtering, and centrifugation is performed at 5000 r / min for enrichment for 5 minutes, the supernatant is discarded, and the precipitate is obtained, and the precipitate is freeze-dried for 72 hours and then packaged for standby use.

[0056] Determination of water content: Place the clean weighing bottle in a drying oven at 120°C for 30 minutes, and weigh W1 after cooling; weigh 1g of spore powder in a weighing bottle and weigh W2, place it in an oven at 120°C for 2 hours, and weigh W3 after natural cooling, and repeat 3 times. Water content (%) = (W3-W1) / (W2-W1)×100. The results are shown in Table 4.

[0057] Determination of spore content: Weigh 10 mg of dried spore powder into a 1.5 mL centrifuge tube, mix evenly with 0.02% Tween-80 solution, use a hemocytometer to determine the spore concentration, and calculate the spore content of the spore powder. The results are shown in Table 4.

[0058] Determination of spore germination rate: Weigh 10 mg of spore powder in a 1.5 mL sterile centrifuge tube and mix evenly with 0.02% Tween-80 solution. Take 1 mL of spore suspension and inoculate it into 20 mL of spore germination solution (spore germination solution formula: sucrose 20 g, peptone 5 g, Tween-80 500 μL, distilled water 1000 mL), shake and mix thoroughly, place in a shaker at 26 ° C, 180 r / min for 24 h, use a hemocytometer to record the number of germinated and ungerminated spores (spore expansion is twice as large as the original spore or spore germination is considered germination), repeat 3 times. Spore germination rate (%) = (germinated spore number / total spore number) × 100, the results are shown in Table 4.

[0059] Table 4 Quality indexes of pure spore powder of Trichoderma harzianum and Psoralea corylifolia

[0060]

[0061] The data in the table are mean ± standard deviation.

[0062] Experimental results: After freeze-drying, the pure spore powder of Trichoderma harzianum YB01 had a water content of 2.9% and a spore content of 3.5×10 11 / g, spore germination rate 95.8%, freeze-dried pure spore powder of Pseudomonas lilacinus PlGZ4903, water content 3.5%, spore content 8.6×10 10 / g, spore germination rate of 91.5%. Combined with the biological control effect of Pseudomonas lilacinus on root-knot nematodes in Example 1, the granule application amount is 0.5g / plant and 10mL of water is applied. When the pure spore powder of Pseudomonas lilacinus PlGZ4903 is added at 0.75%, the spore concentration in water can reach 3.225×10 7 spores / mL, which can reach the concentration required for controlling root-knot nematodes.

[0063] Example 3: Screening and processing of compound granules of Trichoderma harzianum and Psoralea corylifolia

[0064] (1) Vector screening

[0065] Effect of carrier on the mycelial growth of Trichoderma harzianum YB01: Wheat bran, green zeolite, yellow zeolite, white zeolite, yellow wood powder, bamboo powder, poplar wood powder, kaolin, calcium carbonate, attapulgite, diatomite, bentonite, and white carbon black were added to PDA culture medium, and the concentration was 0.03g carrier / mL. After sterilization, pour it into the culture dish, and after the carrier-containing culture medium solidifies, use a 5mm puncher to punch the Trichoderma harzianum cake cultured for 2 days, and place it in a 26℃ incubator for 2-3 days. The control group without adding carrier is used as the control group. When the control group Trichoderma harzianum YB01 bacteria block grows all over the culture dish, the colony diameter of the control group and the treatment group is measured, and after recording the data, the average inhibition rate is calculated according to the formula of the plate confrontation test method. Inhibition rate (%) = [(control group diameter-treatment group diameter) / (control group diameter-bacteria cake diameter)] × 100. SPSS16 software was used to analyze the data, and the results are shown in Table 5.

[0066] Table 5 Effects of different carriers on the mycelial growth of Trichoderma harzianum YB01

[0067]

[0068] The data in the table are mean ± standard deviation. Different letters after the data in the same column indicate significant differences at the P < 0.05 level (DMRT method).

[0069] Experimental results: According to Table 5, wheat bran, bamboo powder, kaolin, diatomaceous earth, and white carbon black have no significant effect on the growth of Trichoderma harzianum hyphae, but can promote its growth. Among them, wheat bran has the strongest ability to promote the growth of Trichoderma harzianum YB01 hyphae, with a colony diameter of 66.70 mm, 2.84 mm longer than the colony diameter of the CK group (63.86 mm). In addition, wheat bran has a low cost, so wheat bran was selected as an inert carrier for granules for subsequent research.

[0070] (2) Adhesive selection

[0071] Effect of adhesive on the mycelial growth of Trichoderma harzianum YB01: Corn starch, soy protein isolate, dextrin, gelatin, and casein were added to PDA culture medium, and the concentration was 0.03g adhesive / mL for testing. After sterilization, it was poured into the culture dish. After the culture medium containing adhesive solidified, the 2-day cultured Trichoderma harzianum YB01 cake was punched with a 5mm puncher and placed in a 26°C incubator for 2-3 days. The control group was taken as the control group without adding carrier, and each treatment was repeated 3 times. When the Trichoderma harzianum block in the control group filled the culture dish, the colony diameter of the control group and the treatment group was measured, and the data was recorded, and the average inhibition rate was calculated according to the formula. Inhibition rate (%) = [(control group diameter-treatment group diameter) / (control group diameter-bacteria cake diameter)] × 100, SPSS16 software was used to analyze the data, and the results are shown in Table 6.

[0072] Table 6 Effects of different adhesives on the growth of Trichoderma harzianum YB01

[0073]

[0074] The data in the table are mean ± standard deviation. Different letters after the data in the same column indicate significant differences at the P < 0.05 level (DMRT method).

[0075] Experimental results: As shown in Table 6, among the five experimental adhesives, dextrin and gelatin had little effect on the mycelial growth of Trichoderma harzianum YB01, and there was no difference in colony diameter compared with CK, with an average inhibition rate of 0.75% and 1.38%. Both dextrin and gelatin can be considered as adhesives for granules for subsequent research and development. Among them, dextrin is cheaper than gelatin, and it will not absorb water and agglomerate during storage, so dextrin is selected as the adhesive for granules for subsequent research and development.

[0076] (3) Screening of disintegrant dosage

[0077] First, take dextrin and hot water and mix them evenly in a ratio of 1:2 to prepare an adhesive for use. Then, the disintegrant xanthan gum is mixed evenly with 2% harzianum YB01 spore powder, 1% lilacinus purpurogenous PlGZ4903 spore powder and the carrier in a ratio of 1%, 2% and 3% respectively. During this period, the adhesive is slowly added, and the total amount of the adhesive is 10%, and the remaining mass is supplemented by the carrier. After kneading and mixing evenly, a soft body is obtained, which is then added to the extrusion granulator for extrusion, and finally placed in an oven at 30°C for 24 hours to obtain a finished granule, and the performance of the granule is recorded. Take 0.5g of the sample and put it into a 250mL beaker containing 100mL of distilled water, stir it slowly with a glass rod, and record the disintegration of the granule in water. The results are shown in Table 7.

[0078] Table 7 Xanthan gum dosage screening

[0079]

[0080] Experimental results: From the results in Table 7, it can be seen that with the increase in the amount of xanthan gum, the strength of the granules is improved. This is because xanthan gum also has a certain viscosity, which can enhance the strength of the granules, but excessive addition will reduce the disintegration performance of the granules. In addition to disintegration, the addition of xanthan gum can have the effect of sustained release and increase the strength of the granules. In summary, the amount of xanthan gum added is selected to be 2%.

[0081] (4) Preparation of Trichoderma harzianum and Psoralea corylifolia Granules

[0082] In terms of percentage, the granules include the following components: 3% spore powder (the mass ratio of pure spore powder of Trichoderma harzianum YB01 to pure spore powder of Pseudomonas lilacinus PlGZ4903 is 2:1), 10% adhesive dextrin, 2% disintegrant xanthan gum and 85% carrier wheat bran.

[0083] The specific preparation method of the granules is as follows: first take 10% dextrin and hot water in a ratio of 1:2 and mix them evenly to prepare a binder for use. Evenly mix 2% disintegrant xanthan gum, 3% spore powder (the mass ratio of pure spore powder of Trichoderma harzianum YB01 and pure spore powder of Pseudomonas lilacinus PlGZ4903 is 2:1) and 85% carrier wheat bran, and then slowly add the binder to the mixture, knead and mix evenly to obtain a soft body, which should be able to form a ball when held and fall apart when touched. Then add it to the extrusion granulator for extrusion, and finally dry it in an oven at 30°C for 24 hours to obtain the finished granule. The finished product is as follows Figure 7 The granules were placed in PDA medium for culture and the results were as follows. Figure 8 As shown, it can be seen that Trichoderma harzianum YB01 and Pseudomonas lilacinus PlGZ4903 can grow normally on the plate.

[0084] Example 4: Application of Trichoderma harzianum and Psoralea corylifolia Granules to Control Cucumber Wilt

[0085] The bacterial cake of cucumber wilt pathogen (F.oxysporum f.sp.cucumerinum) was inoculated into YPD medium and cultured in a shaking incubator at 26°C and 180 r / min for 48 h. After filtering with double-layer gauze, the pathogen spores were enriched by centrifugation and diluted with sterile water to 1.0×10 7 spores / mL spore suspension is ready for use.

[0086] Preparation of granules with different contents of Trichoderma harzianum according to corresponding formulas

[0087] Comparative Example 1

[0088] A composite microbial granule, whose raw materials and preparation method are the same as those in Example 3, the only difference being that the mass ratio of pure spore powder of Trichoderma harzianum YB01 to pure spore powder of Pseudomonas lilacinus PlGZ4903 is 1:5.

[0089] Comparative Example 2

[0090] A composite microbial granule, whose raw materials and preparation method are the same as those in Example 3, the only difference being that the mass ratio of pure spore powder of Trichoderma harzianum YB01 to pure spore powder of Pseudomonas lilacinus PlGZ4903 is 1:4.

[0091] Comparative Example 3

[0092] A composite microbial granule, whose raw materials and preparation method are the same as those in Example 3, the only difference being that the mass ratio of pure spore powder of Trichoderma harzianum YB01 to pure spore powder of Pseudomonas lilacinus PlGZ4903 is 2:3.

[0093] Comparative Example 4

[0094] A composite microbial granule, whose raw materials and preparation method are the same as those in Example 3, the only difference being that the mass ratio of pure spore powder of Trichoderma harzianum YB01 to pure spore powder of Pseudomonas lilacinus PlGZ4903 is 3:4.

[0095] Example 5

[0096] The Trichoderma harzianum and Pseudomonas lilacinus granules include 3% spore powder (the mass ratio of pure spore powder of Trichoderma harzianum YB01 and pure spore powder of Pseudomonas lilacinus PlGZ4903 is 1:1), 10% adhesive gelatin, 1% disintegrant xanthan gum and 86% carrier wheat bran.

[0097] Example 6

[0098] The Trichoderma harzianum and Pseudomonas lilacinus granules include 3% spore powder (the mass ratio of pure spore powder of Trichoderma harzianum YB01 and pure spore powder of Pseudomonas lilacinus PlGZ4903 is 3:1), 10% adhesive gelatin, 3% disintegrant xanthan gum and 84% carrier wheat bran.

[0099] Example 7

[0100] The Trichoderma harzianum and Pseudomonas lilacinus granules include 3% spore powder (the mass ratio of pure spore powder of Trichoderma harzianum YB01 and pure spore powder of Pseudomonas lilacinus PlGZ4903 is 1:2), 10% adhesive gelatin, 2% disintegrant xanthan gum and 85% carrier wheat bran.

[0101] Example 8

[0102] The Trichoderma harzianum and Psoralea lilacinus granules include 3% spore powder (the mass ratio of pure spore powder of Trichoderma harzianum YB01 and pure spore powder of Psoralea lilacinus PlGZ4903 is 1:3), 10% adhesive dextrin, 2% disintegrant xanthan gum and 85% carrier wheat bran.

[0103] Cucumber seeds were grown in seedling trays. After two true leaves grew out, the cucumber seedlings were soaked in the cucumber wilt spore suspension for 30 minutes and then planted in nutrient cups. Granules were applied according to the scheme in Table 8. After each plant was applied, 10 mL of water was added to the nutrient cup. Each treatment was repeated 10 times, with clean water as a control. After 7 days, the growth of cucumbers was observed, the height of cucumber plants was measured, and the disease situation was recorded. The disease index and control effect were calculated according to the following formulas. SPSS16 software was used to analyze the data. The experimental results are shown in Tables 9 and Fig. 9 shown.

[0104] Table 8 Treatment agents and dosage

[0105]

[0106] According to the "Methods of Plant Pathology Research", the classification standards for cucumber seedling wilt are as follows: Level 0: no symptoms; Level 1: the area of ​​yellowing or wilting of true leaves and cotyledons does not exceed 50% of the total area; Level 2: the area of ​​yellowing or wilting of true leaves and cotyledons exceeds 50% of the total area; Level 3: the leaves wilt or die, and only the growth point survives; Level 4: the whole plant wilts severely and dies. Disease index = 100 × [Σ (number of diseased plants × representative level)] / (total number of plants × highest representative value). Control effect (%) = 100 × [(disease index of control group - disease index of treatment group) / disease index of control group].

[0107] Table 9 Effect of granules on cucumber plant height and wilt control effect

[0108]

[0109] The data in the table are mean ± standard deviation. Different letters after the data in the same column indicate significant differences at the P < 0.05 level (DMRT method).

[0110] Experimental results: From Fig. 9As can be seen from Table 9, the disease index of cucumbers without granules was 92.50, the leaves were severely shrunk and yellowed, and the growth was slow; the disease index of cucumbers in each treatment group treated with granules decreased, and the cotyledons also stretched normally. Further analysis of the data in Table 9 shows that the control effect of the granules on cucumber wilt increases with the increase in the proportion of spore powder of Trichoderma harzianum YB01, and the highest control effect can reach 72.97%. Comparing treatment f with treatment h, it is found that the two are consistent in control effect, indicating that the mass ratio of pure spore powder of Trichoderma harzianum YB01 to pure spore powder of Piluginosa PlGZ4903 in the granules is 2:1, and further increasing the proportion of Trichoderma harzianum has no obvious effect on the control effect of cucumber wilt. Comparing treatments i and j with treatments d and e, it is found that treatment i is consistent with treatment d in control effect, and there is no statistically significant difference among the four, indicating that when the mass ratio of pure spore powder is 1:3, there is also a good control effect, and the control effect is above 60%. Comparing treatment j with treatments b and c, it was found that the prevention effect of treatments b and c was significantly reduced. It was speculated that the minimum pure spore powder mass ratio required for the prevention and control of cucumber wilt was 1:3. The insufficient addition of Trichoderma harzianum resulted in poor prevention effect, indicating that the addition of Trichoderma harzianum must reach a certain proportion to have a good prevention effect on cucumber wilt. In addition, the plant height of cucumbers in each treatment group treated with granules was significantly different from that of the clear water control, with the highest being 25.30 mm higher than the clear water control group. The growth-promoting effect on cucumbers also increased with the increase in the proportion of Trichoderma harzianum YB01 spore powder. On the basis of a mass ratio of 2:1 between pure spore powder of Trichoderma harzianum YB01 and pure spore powder of Piluginosa PlGZ4903, further increasing the addition of Trichoderma harzianum spore powder did not significantly increase the plant height of cucumbers. Comprehensive data from each treatment group showed that when the mass ratio of pure spore powder of Trichoderma harzianum YB01 to pure spore powder of Pleurotus eryngii PlGZ4903 in the granules was 2:1, it had a good preventive effect on cucumber wilt and could significantly increase the plant height of cucumber. Further increasing the amount of Trichoderma harzianum added did not significantly improve the preventive effect and plant height. In summary, the mass ratio of pure spore powder of Trichoderma harzianum YB01 to pure spore powder of Pleurotus eryngii PlGZ4903 in the granules was 2:1, which was the most preferred ratio.

[0111] Example 9: Application of 3% Trichoderma harzianum·Purplesporium lilacinum granules on tomatoes

[0112] The test crop was tomato, and the treatments were as follows:

[0113] Treatment group 1: 3% Trichoderma harzianum·Purple spore powder granules (the granule formula is the same as that in Example 3, and the components are as follows in percentage: 3% spore powder (the mass ratio of pure spore powder of Trichoderma harzianum YB01 and pure spore powder of Purple spore powder of PilGZ4903 is 2:1), 10% dextrin, 2% xanthan gum and 85% wheat bran), the application amount is 1500g / mu;

[0114] Treatment group 2: 3% Trichoderma harzianum·Purple spore powder granules (the granule formula is the same as that in Example 3, and the components are as follows in percentage: 3% spore powder (the mass ratio of pure spore powder of Trichoderma harzianum YB01 and pure spore powder of Purple spore powder of PilGZ4903 is 2:1), 10% dextrin, 2% xanthan gum and 85% wheat bran), the application amount is 2500g / mu;

[0115] Treatment group 3: 1% avermectin granules, applied at a rate of 1500 g / mu;

[0116] Treatment group 4: 3% Trichoderma harzianum·Purple spore powder granules (the granule formula is the same as that of comparative example 1, and the components are as follows in percentage: 3% spore powder (the mass ratio of pure spore powder of Trichoderma harzianum YB01 and pure spore powder of Purple spore powder of PilGZ4903 is 1:5), 10% dextrin, 2% xanthan gum and 85% wheat bran), the application amount is 2500g / mu;

[0117] Treatment group 5: 3% Trichoderma harzianum·Purple spore powder granules (the granule formula is the same as that of comparative example 2, and the components are as follows in percentage: 3% spore powder (the mass ratio of pure spore powder of Trichoderma harzianum YB01 and pure spore powder of Purple spore powder of PilGZ4903 is 1:4), 10% dextrin, 2% xanthan gum and 85% wheat bran), the application amount is 2500g / mu;

[0118] Treatment group 6: 3% Trichoderma harzianum·Purple spore powder granules (the granule formula is the same as that of comparative example 3, and the components are as follows in percentage: 3% spore powder (the mass ratio of pure spore powder of Trichoderma harzianum YB01 and pure spore powder of Purple spore powder of PilGZ4903 is 2:3), 10% dextrin, 2% xanthan gum and 85% wheat bran), the application amount is 2500g / mu;

[0119] Treatment group 7: 3% Trichoderma harzianum·Purple spore powder granules (the granule formula is the same as that of comparative example 4, and the components are as follows in percentage: 3% spore powder (the mass ratio of pure spore powder of Trichoderma harzianum YB01 and pure spore powder of Purple spore powder of PilGZ4903 is 3:4), 10% dextrin, 2% xanthan gum and 85% wheat bran), the application amount is 2500g / mu;

[0120] Treatment group 8: No granules were applied, and the amount of water applied was the same as that of the other treatment groups.

[0121] Operation method: Apply the drug 14 days after transplanting the tomatoes. The drug application method is broadcasting. At this time, the height of the tomato plant is about 20cm. Treatment group 8 is the clear water control. Each treatment group is repeated 4 times, and the area of ​​each plot is 50m 2 . A survey was conducted 30 days after the application of the medicine. A five-point sampling method was used. Two plants were surveyed at each point. A total of ten tomato plants were surveyed in each plot. The growth height of the tomatoes was recorded, and the roots were dug out to determine the disease index and calculate the control effect. Disease index

[0122] =100×[Σ(number of disease-grade plants×representative grade)] / (total number of plants×highest representative value). Control effect (%)

[0123] =100×[(control group disease index-treatment group disease index) / control group disease index]. SPSS16 software was used to analyze the data. The test results are shown in Table 10.

[0124] With reference to GB / T 17980.38-2000 Guidelines for Field Efficacy Tests of Pesticides (I) Nematicides for Control of Root-knot Nematode Disease, the grading standards for root-knot nematode disease plants are as follows: Grade 0: no galls on the roots; Grade 1: a small number of small galls on the roots; Grade 3: two-thirds of the roots are covered with small galls; Grade 5: the roots are covered with small galls and have secondary galls; Grade 7: the roots form fibrous root balls.

[0125] Table 10 Effect of granules on tomato plant height and root-knot nematode control

[0126]

[0127] The data in the table are mean ± standard deviation. Different letters after the data in the same column indicate significant differences at the P < 0.05 level (DMRT method).

[0128] Experimental results: The test data in Table 10 show that the disease index of each treatment group decreased after 30 days, and the highest prevention effect can reach 73.64%. Comparing treatment groups 2, 4, 5, 6 and 7, it is found that under the same application rate, there is no significant difference in the prevention effect of granules with different ratios on tomato root-knot nematodes, indicating that when the application rate is 2500g / mu, the mass ratio of pure spore powder of Trichoderma harzianum YB01 and pure spore powder of Piluginosa PlGZ4903 in the granules is 2:1, and there is a good prevention effect. Further increasing the addition amount of pure spore powder of Piluginosa plGZ4903 has no obvious improvement on the prevention effect of root-knot nematodes. The spore production of Piluginosa plGZ4903 is low, and the fermentation substrate rice is generally not reused, so its production cost is higher. On the other hand, the plant height of the treatment group with 3% Trichoderma harzianum·Piluginosa plGZ4903 is higher than that of the clear water control group, indicating that 3% Trichoderma harzianum·Piluginosa granules have a certain growth-promoting effect on tomatoes. There are obvious differences in the effects of different ratios on tomato plant height. The tomato plant height increases with the increase of the proportion of Trichoderma harzianum. The plant height of treatment group 2 is the highest, reaching 94.13 cm. Comparing treatment 1 with treatment 3, treatment 4 and treatment 5, treatment 1 has a smaller application rate but a higher plant height than the other two treatments, indicating that Trichoderma harzianum plays a major role in promoting tomato growth. In summary, all ratios of 3% Trichoderma harzianum·Purple spore granules have a good control effect on tomato root-knot nematodes at a dosage of 2500g / mu. Increasing the proportion of Purple spore has no significant effect on the control effect, but increases the production cost. In addition, the mass ratio of pure spore powder of Trichoderma harzianum YB01 to pure spore powder of Purple spore PlGZ4903 is 2:1, which is the most obvious increase in plant height. Therefore, the mass ratio of pure spore powder of Trichoderma harzianum YB01 to pure spore powder of Purple spore PlGZ4903 is 2:1, which is the most preferred ratio.

Claims

1. A pesticide composition containing Trichoderma harzianum and Pseudomonas lilacinus, characterized in that: The active ingredients of the pesticide composition include pure spore powder of Trichoderma harzianum YB01 and pure spore powder of Pseudomonas lilacinus PlGZ4903; The sum of the mass of the pure spore powder of Trichoderma harzianum YB01 and the pure spore powder of Pseudomonas lilacinus PlGZ4903 is 3% of the total mass of the pesticide composition; The mass ratio of the pure spore powder of Trichoderma harzianum YB01 to the pure spore powder of Pseudomonas lilacinus PlGZ4903 is (1-3):(1-3).

2. The pesticide composition according to claim 1, characterized in that The mass ratio of the pure spore powder of Trichoderma harzianum YB01 to the pure spore powder of Pseudomonas cerevisiae PlGZ4903 is 1:1, 2:1, 3:1, 1:2, and 1:

3.

3. The pesticide composition according to claim 1, characterized in that The mass ratio of the pure spore powder of Trichoderma harzianum YB01 to the pure spore powder of Pseudomonas lilacinus PlGZ4903 is 2:

1.

4. A granule, characterized in that: The granules include the pesticide composition according to any one of claims 1 to 3, and further include adjuvants; the adjuvants include carriers, binders and disintegrants.

5. The granules according to claim 4, characterized in that The granules include the following components in percentage by mass: 0.75-2.25% pure spore powder of Trichoderma harzianum YB01, 0.75-2.25% pure spore powder of Pseudomonas lilacinus PlGZ490, 10% adhesive, 1-3% disintegrant and the remainder of carrier.

6. The granules according to claim 5, characterized in that The granules include the following components in percentage: 2% pure spore powder of Trichoderma harzianum YB01, 1% pure spore powder of Pseudomonas lilacinus PlGZ490, 10% binder, 2% disintegrant and 85% carrier.

7. The granules according to claim 6, characterized in that The carrier comprises at least one of wheat bran, bamboo powder, kaolin, diatomaceous earth and white carbon black, the binder comprises dextrin or gelatin, the disintegrant is xanthan gum, the carrier is wheat bran, and the binder is dextrin.

8. Use of the pesticide composition according to claim 1 or the granules according to any one of claims 3 to 7 in preventing and / or controlling crop wilt or root-knot nematode disease.

9. The use according to claim 8, characterized in that The crop wilt disease is cucumber wilt.

10. The use according to claim 8, characterized in that The root knot nematode disease is tomato root knot nematode disease.

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