Application of Trichoderma asperellum SFC-3 in degrading carfentrazone-ethyl

Abstract

The application of a strain of Trichoderma asperellum SFC-3 in degrading carfentrazone-ethyl. The Trichoderma asperellum SFC-3 is capable of growing by using carfentrazone-ethyl as the sole carbon and nitrogen source and degrading carfentrazone-ethyl. The present application provides the fungal strain Trichoderma asperellum SFC-3 with high-efficiency degradation effect on carfentrazone-ethyl for the first time. The strain is capable of growing by using carfentrazone-ethyl as the sole carbon and nitrogen source, has a wide degrading environmental temperature (the degrading capacity is the strongest at 25 DEG C, and the strain can also grow at 40 DEG C), and a wide pH value (the degrading effect is the best at a pH value of 8, and the strain also has better degrading capacity within the pH value range of 6-10). Meanwhile, the strain has simple culture conditions, is easy to be industrialized, and has significant social and economic benefits.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environmental remediation microorganism technology, and particularly to a Trichoderma asperellum SFC-3 and application thereof in degrading topramezone. BACKGROUND

[0002] Pesticides are currently one of the effective technical means to ensure agricultural production. With the increasing demand for food, modern agriculture relies more and more on synthetic chemical pesticides such as herbicides, insecticides and fungicides for the prevention and control of pests and diseases and the regulation of plant growth. Since the 1980s, the use of herbicides in China has been increasing in quantity and area. The application of herbicides has improved the efficiency of agricultural production, ensured crop yield, guaranteed food supply and brought great benefits to mankind. However, long-term and large-scale use of herbicides can cause serious harm to soil, water, plants and animals.

[0003] Corn is one of the important food crops in the world. In order to prevent weeds from causing yield reduction and quality decline of corn, various herbicides have been developed. Topramezone, also known as benzofurazanone, is a new type of high selective benzyl pyrazolone herbicide developed by BASF Company, which has broad-spectrum weed killing activity, long duration and strong compatibility, and can effectively control annual grasses and broadleaf weeds in corn fields. However, topramezone is difficult to degrade in natural conditions and belongs to long-residual herbicide. Large-scale application of topramezone can cause soil residue and other pesticide hazards, and the harm to biology and environment cannot be ignored. Studies have shown that topramezone inhibits 4-HPPD (p-hydroxyphenyl pyruvate dioxygenase) in mice, leading to an increase in tyrosine levels in serum, and causing adverse effects on eyes, liver, kidney, pancreas and thyroid. Further toxicological studies have shown that topramezone can increase the risk of changes and variations of bone mineralization sites in mice and rabbits. Another study has shown that topramezone can increase the risk of thyroid tumors in mice. Therefore, unreasonable use of topramezone can cause potential harm to human health and the environment. In addition, the soil of corn fields treated with topramezone has a significant inhibitory effect on the following sensitive crops such as rice. At present, researchers at home and abroad mainly study the control effect of topramezone on weeds, lead derivatives, crop impact, and photolysis, hydrolysis and soil adsorption and residue, and there is no report on the biodegradation and degradation characteristics of topramezone. SUMMARY

[0004] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present application is to provide a Trichoderma asperellum SFC-3 and application thereof in degrading topramezone, which can effectively solve the problem that there is no report on the biodegradation and degradation characteristics of topramezone.

[0005] In order to achieve the above-mentioned purpose, the technical scheme solved by the present application is an application of a Trichoderma asperellum SFC-3 in degrading carfentrazone-ethyl.

[0006] The Trichoderma asperellum SFC-3 grows by using carfentrazone-ethyl as the only carbon and nitrogen source and degrades carfentrazone-ethyl.

[0007] Further, the Trichoderma asperellum SFC-3 is classified and named as Trichoderma asperellum (T. asperellum) SFC-3. Trichoderma asperellum It has been preserved in the China General Microbiological Culture Collection Center, the preservation date is August 22, 2018, the preservation number is CGMCC No. 16097, and the preservation address is No. 1, Beichen Road, Chaoyang District, Beijing.

[0008] The present application first provides the fungal strain Trichoderma asperellum SFC-3 which has a high-efficiency degradation effect on carfentrazone-ethyl. The strain can grow by using carfentrazone-ethyl as the only carbon and nitrogen source, has a wide degradation environmental temperature and pH value, and meanwhile, the strain culture condition is simple, easy for industrial production, and has significant social and economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 is an influence diagram of additional carbon and nitrogen sources on the degradation of carfentrazone-ethyl by the strain SFC-3 in the present application.

[0010] Figure 2 is an influence diagram of temperature on the degradation of carfentrazone-ethyl by the strain SFC-3 in the present application.

[0011] Figure 3 is an influence of pH on the degradation of carfentrazone-ethyl by the strain SFC-3 in the present application.

[0012] Figure 4 is a carfentrazone-ethyl residual amount diagram in different treatment soils in the present application. DETAILED DESCRIPTION

[0013] The specific embodiments of the present application are described in detail below in combination with the drawings and specific cases.

[0014] The Trichoderma asperellum SFC-3 in the present application is isolated from the rhizosphere soil of vegetables in Liuxian County, Anyang City, Henan Province. The strain SFC-3 is cultured on PDA solid culture medium, the colony is white in the early stage, the center is light green, the aerial hypha is fine and woolly, is scattered from the center to the periphery, the back of the colony is colorless, the colony gradually changes to light green in the middle stage, the center is dark green, the aerial hypha changes to cotton-like, and the whole colony is dark green in the late stage. The observation under a microscope shows that the conidium is green, and the shape is spherical or ellipsoidal.

[0015] BLAST analysis of the ITS sequence of SFC-3 with sequences in GenBank revealed that *Trichoderma hygroscopicum* had the highest similarity to SFC-3 strain. Trichoderma asperellum The homology reached 99%. A phylogenetic tree was constructed using similar type strains. The constructed phylogenetic tree shows that SFC-3 is closely related to *Trichoderma echinosporum* (…). Trichoderma asperellum They belong to the same genetic branch. Based on their morphological characteristics, strain SFC-3 was ultimately identified as *Trichoderma echinosporum*. Trichoderma asperellum This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 16097.

[0016] The *Trichoderma echinococcus* strain SFC-3 of this invention can grow using benzoxazole as the sole carbon and nitrogen source and degrade benzoxazole. It exhibits a wide degradation range with suitable temperature (20-40℃) and pH (6-10), demonstrating good degradation efficiency. Relevant information is as follows:

[0017] I. Determination of the degradation ability of Trichoderma echinosporum SFC-3 on benzoxazine

[0018] Five-mm diameter *Trichoderma echinococcus* SFC-3 mycelial cakes were inoculated into an inorganic salt medium containing 100 mg / L benzoyl permethrin (NaCl 1.0 g / L, K₂HPO₄ 1.5 g / L, KH₂PO₄ 0.5 g / L, MgSO₄ 0.5 g / L), with 1 g / L NH₄NO₃ added as the sole nitrogen source. The medium was incubated at 30 °C and 180 r / min. The treatment was repeated three times, with uninoculated samples serving as controls. After 7 days of incubation, two volumes of dichloromethane were added to the culture medium, and the mixture was shaken for 30 min at 30 °C. The mixture was then allowed to stand, the supernatant was discarded, and the lower layer was evaporated to dryness using a rotary evaporator. The residue was dissolved in chromatographic methanol, filtered through an organic phase microporous membrane (0.22 µm), and then analyzed by high-performance liquid chromatography (HPLC). High performance liquid chromatography (HPLC) detection conditions for benzoxazine: Column: 4.6 mm * 250 mm, ZORBAX Extend-C18; mobile phase: methanol:water (ammonium acetate 1.25 g / L) = 20:80 (V:V); column temperature: 30°C; flow rate: 1.0 mL / min; detection wavelength: 255 nm.

[0019] Degradation rate (%) = (Control benzoxazole concentration - Treatment benzoxazole concentration) / Control benzoxazole concentration

[0020] The test results showed that Trichoderma hygroscopicum SFC-3 had a degradation rate of 52.37% for benzoxazine, indicating a good degradation effect.

[0021] II. The effect of additional carbon and nitrogen sources on the degradation of carfentrazone-ethyl by strain SFC-3

[0022] In order to explore the effect of additional carbon and nitrogen sources on the degradation of carfentrazone-ethyl by strain SFC-3, four different treatments were set up, i.e. 3 g / L glucose was added as an additional carbon source (TOP+C) in the inorganic salt medium containing 100 mg / L carfentrazone-ethyl; 1 g / L NH4NO3 was added as an additional nitrogen source (TOP+N); 3 g / L glucose and 1 g / L NH4NO3 were added as additional carbon and nitrogen sources (TOP+CN); and no additional carbon and nitrogen sources (TOP). The SFC-3 mycelium disks with a diameter of 5 mm were inoculated into the above different treatment media and cultured at 30°C and 180 r / min. Each treatment was repeated three times, and un-inoculated samples were used as controls. After 7 days, the residual carfentrazone-ethyl concentration in the medium was detected by high performance liquid chromatography, and the degradation rate was calculated.

[0023] The results showed that Figure 1 the treatment group with only carfentrazone-ethyl as the sole carbon and nitrogen source (TOP) had the highest degradation rate, and the addition of carbon (glucose) and nitrogen (NH4NO3) sources decreased the degradation effect of carfentrazone-ethyl. This indicated that SFC-3 could grow using carfentrazone-ethyl as the sole carbon and nitrogen source, and the additional carbon and nitrogen sources affected the degradation effect of SFC-3 on carfentrazone-ethyl.

[0024] III. The effect of environmental factors on the degradation of carfentrazone-ethyl by strain SFC-3

[0025] In order to study the effects of different environmental factors such as pH and temperature on the degradation of carfentrazone-ethyl by strain SFC-3, different temperature (10, 20, 25, 30, 35, and 40°C) and pH (4, 5, 6, 7, 8, 9, and 10) treatment groups were set up. The SFC-3 mycelium disks with a diameter of 5 mm were inoculated into the inorganic salt medium containing 100 mg / L carfentrazone-ethyl as the sole carbon and nitrogen source, and all samples were treated according to the above different pH and temperature and then shaken. Un-inoculated samples were used as blank controls, and each treatment group had three replicates. After 7 days, the residual carfentrazone-ethyl concentration in the medium was detected by high performance liquid chromatography, and the degradation rate was calculated.

[0026] The results showed that Figure 2 and Figure 3 the degradation effect of strain SFC-3 on carfentrazone-ethyl was the best at 25°C and pH 8.

[0027] IV. The remediation ability of strain SFC-3 on carfentrazone-ethyl contaminated soil

[0028] The prepared air-dried soil was sieved to prepare the contaminated soil containing carfentrazone-ethyl at a concentration of 15 mg / kg. The Trichoderma asperellum SFC-3 spore solution was inoculated into the soil at an inoculation amount of 10%. The soil sample without inoculation was used as a control, and each treatment had 3 replicates. All samples were placed in a 25℃ environment and watered regularly to maintain the soil moisture content. The carfentrazone-ethyl content in the soil of different treatments was determined on the 3rd, 7th, 10th, and 15th day of cultivation, respectively.

[0029] The results are shown in Table 1. Figure 4 As can be seen from Table 1, on the 3rd day of cultivation, there was a large difference in the concentration of carfentrazone-ethyl between the inoculated and non-inoculated soil samples. As the cultivation time increased, the difference in the concentration of carfentrazone-ethyl between the inoculated and non-inoculated soil samples gradually increased. After 15 days of simulated remediation, the residual amount of carfentrazone-ethyl in the non-inoculated soil sample was 12.45 mg / kg, while no residual carfentrazone-ethyl was detected in the inoculated soil sample. This indicates that the strain SFC-3 has good ability to degrade carfentrazone-ethyl in soil.

[0030] As can be seen from the above, compared with the prior art, the application provides the use of the Trichoderma asperellum SFC-3 strain in the degradation of carfentrazone-ethyl, which has a high-efficiency degradation effect on carfentrazone-ethyl. The strain can grow using carfentrazone-ethyl as the sole carbon and nitrogen source, has a wide range of environmental temperature for degradation (the degradation ability is strongest at 25℃, and the strain can also grow at 40℃), and has a wide range of pH values (the degradation effect is best at a pH value of 8, and the strain also has good degradation ability at a pH value of 6-10). This is a great innovation in the biological degradation of carfentrazone-ethyl. The strain has simple cultivation conditions, is easy to produce industrially, has good development and application prospects, and has significant social and economic benefits.

[0031] It should be pointed out that the above is only a preferred embodiment of the present application, and does not limit the present application in any form. Any person skilled in the art can make changes or modifications to the equivalent embodiments within the scope of the technical solutions of the present application without departing from the scope of the present application, which are all within the protection scope of the present application.

Claims

1. Application of a strain of Trichoderma asperellum SFC-3 in degrading carfentrazone-ethyl, the strain utilizes carfentrazone-ethyl as the sole carbon and nitrogen source for growth and degrades carfentrazone-ethyl, the degradation temperature is 25℃, the pH value is 8, and the biological preservation number is CGMCC No. 16097.

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