CN116083244A - Aspergillus terreus DT1, microbial inoculum, and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of microorganism and environmental protection, and particularly relates to aspergillus terreus DT1, a microbial inoculum, a preparation method and application thereof. Aspergillus terreus (Aspergillus terreus) DT1 is preserved in China general microbiological culture Collection center (CGMCC) for 09 and 15 days in 2021, the preservation number is CGMCC No.23231, the Aspergillus terreus DT1 can efficiently degrade nicosulfuron, and the degradation rate of the strain to the nicosulfuron can reach 98.05% at the temperature of 30 ℃ and the pH value of 5.8 for 119 hours, thereby laying a theoretical foundation for the strain to be applied to soil remediation polluted by the nicosulfuron.

Description

Aspergillus terreus DT1, microbial inoculum, and preparation method and application thereof

Technical Field

The invention belongs to the field of microorganism and environmental protection, and particularly relates to aspergillus terreus DT1, a microbial inoculum, a preparation method and application thereof.

Background

Nicosulfuron (nicosulfuron) is widely used in corn fields as a sulfonylurea herbicide because of the advantages of safety, high efficiency, low dosage and the like, and has remarkable control effect on annual weeds and broadleaf weeds. However, long-term large-scale use not only causes the problems of soil pesticide residue, weed drug resistance and the like, but also can cause serious influence on wheat, sorghum, potatoes and the like of the aftercrop sensitive crops, thereby resulting in yield reduction and harvest failure. Therefore, the problem of nicosulfuron residue has become a soil pollution problem which is currently urgently needed to be solved.

The utilization of microbial degradation has become an important means for solving the residual problem of nicosulfuron at present. At present, research on microbial degradation of nicosulfuron is gradually increased at home and abroad, and various degradation strains are separated and identified, and mainly comprise fungi, bacteria and actinomycetes. Carles L et al separate a Pseudomonas fluorescens SG-1 with a degradation rate of 77.5% and a bacterial Mes11 from the nicosulfuron-rich soil, the bacterial strain has high degradation activity, and can degrade various sulfonylurea herbicides at the same time, and the degradation characteristics and metabolic products of the sulfonylurea herbicides are studied. Zhang Guomin and the like, 1 strain of photosynthetic bacteria J5-2 for efficiently degrading nicosulfuron is isolated from corn fields using nicosulfuron for a long period of time, the strain is identified as rhodopseudomonas and is cultured for 7d at a temperature of 30 ℃ and a pH of 7.0, and the strain is specific to 400 mg.L ^-1 The degradation rate of the nicosulfuron is 32.2 percent. Zhou Shan et al screened a strain LAM-WHM-ZC from marine sediments that efficiently degraded nicosulfuron, identified as Oceanisophaera genus, which was isolated from 50 mg.L in 8d ^-1 The degradation rate of the nicosulfuron reaches 83.8 percent. Most strains screened at present have low degradation rate on nicosulfuron and cannot be practically applied.

Disclosure of Invention

The invention aims to provide aspergillus terreus DT1, a microbial inoculum, a preparation method and application thereof aiming at practical problems and demands in production practice.

In a first aspect of the present invention, there is provided aspergillus terreus (Aspergillus terreus) DT1 having a accession number of CGMCC No.23231.

In a second aspect of the invention, the application of the aspergillus terreus DT1 in degrading nicosulfuron is provided.

In a third aspect of the invention, a microbial inoculum comprising the aspergillus terreus DT1 is provided.

In a fourth aspect of the present invention, there is provided a method for preparing the microbial inoculum, comprising the steps of:

s1, inoculating the aspergillus terreus DT1 into a PDB liquid culture medium for culturing for 48-72 hours, and regulating the OD of a bacterial liquid ₆₀₀ A value of 1.0 as seed liquid;

s2, inoculating the seed solution into a PDB liquid culture medium according to the inoculation amount of 2%, and culturing for 48-144h in a shaking table at 30 ℃ and 160r/min, wherein the cultured culture solution is the microbial inoculum.

In a fourth aspect, the invention provides an application of the microbial inoculum in degrading nicosulfuron.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Preservation description:

strain name: aspergillus terreus;

latin name: aspergillus terreus;

strain number: DT1;

preservation mechanism: china general microbiological culture Collection center (CGMCC);

address: the institute of microorganisms of national academy of sciences of China, national institute of sciences, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing;

preservation date: 2021, 09, 15;

preservation number: CGMCC No.23231.

Drawings

FIG. 1 is a colony characterization of A.terreus DT 1.

FIG. 2 is a system development tree of A.terreus DT1 constructed based on 16S rRNA.

FIG. 3 is a graph showing the relationship between the growth rate of A.terreus DT1 and the degradation rate of nicosulfuron.

FIG. 4 shows the effect of temperature, pH, inoculum size and substrate concentration on the growth and degradation rate of A.terreus DT 1.

FIG. 5 shows the results of RDA analysis between different factors and degradation rates.

FIG. 6 is a plot of the response of nicosulfuron degradation rate (time, pH and temperature).

Detailed Description

The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.

Example 1: isolation and characterization of aspergillus terreus DT1

1 materials and methods

1.1 reagents for test

95% nicosulfuron technical (supplied by Beijing Soy Bao technology Co.).

1.2 test soil

Corn field soil for applying nicosulfuron in Dazhou city of Shanxi province for a long period.

1.3 test Medium

Referring to the prior art (Ji Meng, zhao, dong Jingao, etc.. Screening of degraded nicosulfuron strains and the degradation characteristics of the degraded nicosulfuron were initially discovered [ J ]. Ind. Agrochemical report, 2014, 16 (3): 330-336.), the culture medium formulation was prepared into a PDB culture medium, an inorganic salt culture medium and a basic salt culture medium.

2 test method

2.1 enrichment isolation and identification of degraded nicosulfuron Strain

1.0g of the collected soil sample is weighed in 100mL of inorganic salt culture medium (the concentration of the nicosulfuron is 100 mg.L) ^-1 ) At 30℃and 160 r.min ^-1 After 7d of culture in the shaking table, the high-concentration nicosulfuron is transferred into the high-concentration nicosulfuron according to the inoculation amount of 10 percentIn the organic salt culture medium, the maximum concentration of the nicosulfuron is 400 mg.L ^-1 . Absorbing 1mL of enrichment solution to dilute 7 gradients of 10 respectively ^-2 -10 ^-8 Uniformly coating 0.5mL of different gradient dilutions to obtain nicosulfuron with the content of 400 mg.L ^-1 Culturing at 30 deg.c for 24-120 hr, separating and purifying with single bacterial colony with "hydrolysis ring" and high performance liquid chromatography to detect the nicosulfuron degrading effect. And selecting the strain DT1 with highest degradation efficiency, observing colony morphology, and sending to Hua big gene company for 16S rRNA sequencing to construct a phylogenetic tree.

3 results

1 strain of nicosulfuron high-efficiency degradation strain is obtained by screening and separating the collected soil enrichment domestication, and is named as DT1, the strain is inoculated on a PDA solid culture medium for culture, white colonies are formed after 2 days, and the bacterial colonies are observed to have hyphae with more branches and are yellow green under a microscope; after 4d the colonies produced a large number of spores of a tan; after 7d of culture, the diameter of the colony is 3.0-3.5cm, and the color of the colony is earthy yellow (see FIG. 1).

Sequencing by 16S rRNA, and comparing the homology of the sequencing result of the fragment with the nucleic acid sequence in NCBI database by using a Blastn program, the bacterial strain DT1 and Aspergillus terreus have the closest parent relationship. Further combining with the morphological and sequence characteristics of the strain DT1, this strain was identified as Aspergillus terreus (Aspergillus terreus), and its phylogenetic tree is shown in FIG. 2.

Example 2: degradation of nicosulfuron by aspergillus terreus DT1

1 test method

1.1 determination of degradation Curve and growth Curve of Strain DT1

Inoculating the bacterial strain DT1 into a PDB liquid culture medium for culturing for 48-72 hours, and regulating the bacterial liquid OD ₆₀₀ The value was 1.0 as a seed solution. Inoculating the seed solution into 50mL PDB liquid culture medium according to 2% inoculum size, repeating for 3 times, and standing at 30deg.C for 160r.min ^-1 Culturing in a shaking table for 144h, sampling every 24h, drying, and measuring the dry weight of the bacterial body. Under the same conditions, the concentration of the added solution is 100 mg.L ^-1 Is measured, and the residual concentration of nicosulfuron (ginsengAccording to Polati S, bottaro M, frascarolo P, et al HPLC-UV and HPLC-MSn multiresidue determination of amido-sulforon, azimsulfuron, nicosulfuron, rimmsulfuron, thifensulfuron methyl, trbenuron methyl and azoxys-trobin in surface waters [ J ]]Analyt Chim Acta,2006,579 (2): 146-151) and calculate the degradation rate.

Nicosulfuron degradation rate% = (control nicosulfuron mass concentration-treatment nicosulfuron mass concentration)/control nicosulfuron mass concentration.

1.2 effects of temperature, pH, inoculum size and substrate concentration on growth of degradation bacteria DT1 and degradation rate of nicosulfuron

The strain DT1 was inoculated to a strain containing 100 mg.L ^-1 Four groups of tests are set in a PDB liquid culture medium of nicosulfuron, and the temperatures are respectively set to be 20, 25, 30, 35 and 40 ℃; adjusting the pH to be 5.0, 6.0, 7.0, 8.0 and 9.0 respectively; inoculating at 1%, 2%, 4%, 5%, 6%, and setting substrate concentration at 100, 200, 300, 400, 500 mg.L at 2% ^-1 The method comprises the steps of carrying out a first treatment on the surface of the The four groups of tests are placed at 30 ℃ and 160 r.min ^-1 Culturing on a shaking table for 120h, repeating for 3 times by taking non-inoculated bacterial liquid as a control, measuring the residual concentration of nicosulfuron by using a high performance liquid chromatography mass spectrometer, and calculating the degradation rate; the strain growth was also determined.

1.3 screening of Critical factors for degradation of nicosulfuron by Strain DT1 based on RDA analysis

RDA analysis was performed on single factor test results using Canoco 5.0 software to determine key factors affecting strain degradation rate.

1.4 response surface method optimization of degradation conditions of Strain DT1

In order to further optimize key influencing factors of the strain DT1 for degrading the nicosulfuron, the optimal conditions of the strain for degrading the nicosulfuron are obtained. Box-Behnken test Design (see Table 1) was performed using Design expert8.0.6 software with time (24-144 h), temperature (20-40 ℃) and pH (5-9) as influencing factors and degradation rate as a response value, respectively.

Table 1 three factor test design

2 results

2.1 relation between growth Rate and degradation Rate of Strain DT1

From fig. 3, it can be obtained that: the growth of the strain DT1 has positive correlation with the rate of nicosulfan Long Jiangjie. In the presence of the initial concentration of 100 mg.L ^-1 In an inorganic salt culture medium of the nicosulfuron, after the culture for 24 hours, the growth rate of the strain is slow growth phase, and the degradation rate of the nicosulfuron is slow; the culture time is 48-120h, the growth rate of the strain is the fastest in the logarithmic growth phase, the degradation rate of nicosulfuron is faster, and the degradation rate reaches 93.87% in 120 h; the growth rate of the strain is gradually reduced after 120 hours of culture, the growth amount of thalli reaches the maximum, the growth enters a stabilization period, the degradation rate of nicosulfuron is less changed, and the strain tends to be stable; after 144 hours of culture, the strain starts to grow and has a descending trend, enters into the decay period, and has no obvious change on the degradation rate of nicosulfuron.

2.2 study of optimal conditions for degrading nicosulfuron by the Strain DT1

From fig. 4, it can be obtained that: the degradation effect of the bacterial strain DT1 on the nicosulfuron is obviously different under the influence of temperature, and the growth rate of the bacterial strain and the degradation efficiency of the nicosulfuron are in a trend of rising and then falling along with the rising of the temperature; the strain can grow normally within the range of 30-35 ℃, the growth amount of the strain is highest at 30 ℃, and the degradation rate of the nicosulfuron is up to 93.08%. When the PH value is in the range of 5.0-9.0, the growth and metabolism of the strain are normal, the efficiency of degrading the nicosulfuron is over 80%, and the growth of the strain and the degradation rate of the nicosulfuron under the acidic condition are obviously higher than those under the alkaline condition; when the PH value is 5, the growth amount of the strain is maximum, and the degradation rate of the nicosulfuron reaches 92.9 percent. The growth of the strain increased significantly with the increase of the inoculum size, but the degradation rate difference of different inoculum sizes was not significant, and the degradation rates of 1%, 2%, 4%, 5% and 6% of the inoculum sizes were 90.8%, 90.9%, 91.2%, 92.2% and 92.3%, respectively. The initial concentration of nicosulfuron is obviously different from the degradation rate and the growth curve of the strain DT1, and the degradation rate and the growth amount are in a trend of increasing and then decreasing. The initial concentration is 100 mg.L ^-1 The highest degradation rate of the strain was 93.35The corresponding strain growth is the largest; when the initial concentration is 500 mg.L ^-1 When the strain is degraded, the degradation capacity and the growth amount are obviously reduced.

From fig. 5, it can be obtained that: the time, the temperature, the inoculation amount and the substrate concentration are obtained through redundant analysis with the degradation rate of nicosulfuron, the included angle between the time, the temperature, the inoculation amount and the degradation rate is an acute angle, which shows that the two factors are positively correlated with the degradation rate, and the PH value and the substrate concentration are obtuse angles, which shows that the three factors are negatively correlated with the degradation rate. The inoculation amount and the substrate concentration are short according to the line length, which shows that the influence of the inoculation amount and the substrate concentration on the degradation rate is small; it can be seen from RDA analysis that time, pH and temperature are the main factors affecting the degradation rate of the strain.

2.3 response surface method optimizing degradation conditions of bacterial strain DT1 on nicosulfuron

The interaction of the bacterial strain DT1 for degrading the nicosulfuron is analyzed by using Design Expert 13.0, wherein the interaction is influenced by three factors (table 2) of time, temperature and PH, and a square polynomial regression equation of the degradation rate of the nicosulfuron, the time, the temperature and the PH value is established by using a Box-Behnken Design result to carry out variance analysis: degradation rate%= +91.03+31.45×a-2.31×b+1.56×c+0.16×ab-0.1725×ac-0.2050×bc-31.55×a ² -5.83*B ² -5.64*C ² +1.45*A ² B. From Table 3, the model demonstrates that the effect of different growth conditions on bacterial strain DT1 degradation of nicosulfuron was significant for all 3 dependent variables, and A, B, C, A in the analysis of variance ² 、B ² And C ² All reach significant level, which means that the time, temperature and PH have larger influence on the degradation rate of the strain for degrading the nicosulfuron; the model mismatch in the table is not significant, which indicates that the model is suitable for analyzing the influence condition of the strain degradation rate, and can be used for predicting the optimal adaptation condition of the strain. The optimal degradation condition optimization of the strain is carried out by using the model under the limiting conditions of 96-120h, 5-7 PH value, 28-32 ℃ and 90% -95% degradation rate, and the degradation rate of the strain DT1 can reach 98% at 119h, 5.9 PH value and 30 ℃. After verification of the degradation rate of the strain under the condition, the degradation rate of the strain is found to be 98.05%.

TABLE 2 results of the design test of the degradation rate response surface method for strain DT1

TABLE 3 analysis of variance table (type III square sum of squares)

As can be seen from fig. 6: when the PH is fixed, the degradation rate of the strain to the nicosulfuron is stable after increasing along with the increase of time, and the strain tends to be increased and then reduced along with the increase of temperature; when the temperature is fixed, the degradation rate tends to be stable after increasing along with the increase of time, and the degradation rate tends to be increased firstly and then reduced along with the increase of the temperature; the degradation rate tends to increase and decrease with increasing pH and temperature over time. From the response surface map and analysis of variance results, it follows: the effect of 3 factors on degradation rate is as follows: the time is more than PH temperature, the interaction of time with PH and time with temperature is larger, and the degree of change of the curved surface of the response surface is large; and the interaction effect of PH and temperature is smaller, and the degree of change of the curved surface of the response surface is small.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (5)

1. Aspergillus terreus (Aspergillus terreus) DT1 with a preservation number of CGMCC No.23231.

2. The use of aspergillus terreus DT1 as claimed in claim 1 for degrading nicosulfuron.

3. A microbial inoculum comprising the Aspergillus terreus DT1 of claim 1.

4. A method for preparing the microbial inoculum of claim 3, comprising the steps of:

s1, inoculating the aspergillus terreus DT1 into a PDB liquid culture medium for culturing for 48-72 hours, and regulating the OD of a bacterial liquid ₆₀₀ A value of 1.0 as seed liquid;

s2, inoculating the seed solution into a PDB liquid culture medium according to the inoculation amount of 2%, and culturing for 48-144h in a shaking table at 30 ℃ and 160r/min, wherein the cultured culture solution is the microbial inoculum.

5. The use of the microbial inoculum of claim 3 for degrading nicosulfuron.

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