CN113308380B - Antibacterial activity of trichoderma and application thereof - Google Patents
Antibacterial activity of trichoderma and application thereof Download PDFInfo
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- CN113308380B CN113308380B CN202110429251.1A CN202110429251A CN113308380B CN 113308380 B CN113308380 B CN 113308380B CN 202110429251 A CN202110429251 A CN 202110429251A CN 113308380 B CN113308380 B CN 113308380B
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2442—Chitinase (3.2.1.14)
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01014—Chitinase (3.2.1.14)
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Abstract
The invention discloses an antibacterial activity of trichoderma aureoviride and application thereof, the trichoderma aureoviride is Trichoderma aureoviride QTYC44 with a preservation number of CCTCC M2014267 preserved by China center for type culture preservation. The Trichoderma aureoviride has good bacteriostatic action on cucumber fusarium wilt bacteria, wheat gibberella and tobacco brown spot bacteria, has good prevention and treatment effect on cucumber fusarium wilt, has good chitinase activity, and can be used for preparing a biocontrol microbial inoculum to prevent and treat fungal diseases.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an application of an antibacterial activity of Trichoderma sp.
Background
Trichoderma (Trichoderma spp.) fungi are classified in Deuteromycotina, class Hyphomycetes, order Hyphomycetales, family Conidiobolus, genus Trichoderma. Trichoderma fungi are abundant in species and widely exist in soil, plant surroundings and other environments. The trichoderma is a kind of multifunctional environment-friendly fungi, and is widely applied to the fields of agriculture, plant protection, environmental protection and the like.
The trichoderma is from yellow dragonfish larva intestinal tract, the insect intestinal tract is the most important colonization site of microorganisms, a series of characteristics of the insect intestinal tract are beneficial to the colonization of the microorganisms, including the colonization of the microorganisms related to food intake, the nutrient availability of the intestinal tract is strong, and the pressure (such as drying, ultraviolet rays and the like) of the external environment is avoided. Different insect gut morphologies and physicochemical properties vary widely, and these factors have a large impact on microbial community architecture. The intestinal symbiotic bacteria have the unique characteristics of closely symbiotic with host insects and long-term natural selection and co-evolution
In recent years, the use of chemical pesticides in large quantities has caused environmental pollution and drug resistance of pathogenic bacteria. In order to avoid the health threat of human beings, the search for drug source compounds with new structural characteristics or new action characteristics becomes a main method for coping with the rapid change of pathogenic bacteria. The insect symbiotic bacteria have special metabolic pathways in the long-term evolution process with the host, and are valuable resource libraries for the research and development of novel biological pesticides. Therefore, the isolation of antibacterial active substances with novel structures from insect intestinal symbiotic bacteria is one of the means of green agriculture at present.
Disclosure of Invention
Provided herein are uses of the antibacterial activity of Trichoderma sp.
The Trichoderma (Trichoderma sp.) QTYC44 strain is preserved in China center for type culture Collection with the preservation number: CCTCC M2014267, address: eight-way No. 299 in Wuchang area of Wuhan city, Hubei province.
The basic culture form of Trichoderma sp.QTYC44 is as follows: after the bacterial strain QTYC44 is cultured on a PDA plate for three days at 25 ℃, the diameter of a bacterial colony is larger than 65mm, the bacterial colony completely covers a culture dish, the optimal growth temperature is 25-36 ℃, light yellow green pigment is generated on the culture medium, and the bacterial colony turns green after 7 days. Has no obvious smell and no diffused pigment. Hyphae are rare and conidia form after 3 days. First, fine branches are generated, and then, light green fine particles are grown. Conidiophores are colorless, secondary branches are formed, and meristematic cells are specialized into bottleneck shapes. Conidiophores are monosporary, colorless, round to oval, 3-4X 2-2.5 μm. The hyphae were colorless with septa and about 1.5-3 μm wide. Chlamydospores appeared after 2 weeks, terminal, smooth, spherical or dumbbell shaped, 6-6.5X 5-6 μm.
The invention provides an antibacterial active component chitinase of Trichoderma sp.
The invention provides an antibacterial activity application of Trichoderma sp.
The culture medium has the following formula
PDA/PD: 200.0g of potato, 20.0g of glucose, 15-20g of agar powder and 1L of distilled water. No agar powder is added to be used as a PD liquid culture medium.
MYG: 10g of glucose, 5g of maltose, 5g of yeast extract and 1L of distilled water.
Chitinase induction medium: 3g of ammonium nitrate, 3g of monopotassium phosphate, 0.65g of magnesium sulfate, 0.15g of ferrous sulfate, 9g of colloidal chitin and distilled water until the volume is 1L.
The preparation method of the colloidal chitin in the chitinase induction culture medium comprises the following steps: weighing 12g of chitin powder, pouring into a triangular flask, slowly pouring 100mL of concentrated hydrochloric acid into the triangular flask in a ventilated kitchen, placing the triangular flask in an ice box for cooling, stirring for 30min by using a glass rod, standing for 24h in a refrigerator at 4 ℃, adding the sample into 3L of sterile water, standing for 12h in the refrigerator at 4 ℃, adjusting the pH value to 7 by using hydrochloric acid and sodium hydroxide, filtering out supernate by using a Bush funnel, and cleaning for three times by using sterile water to obtain about 9g of colloidal chitin.
The invention has the following beneficial effects:
1. the invention separates and obtains the strain Trichoderma QTYC44 for the first time.
2. The fermentation liquor of the Trichoderma strain QTYC44 provided by the invention has better chitinase activity.
3. The Trichoderma QTYC44 strain provided by the invention has good antagonistic action on a plurality of plant pathogenic bacteria such as cucumber fusarium wilt, wheat gibberella, tobacco alternaria alternata and the like.
4. The Trichoderma QTYC44 strain provided by the invention has good prevention and treatment effects on cucumber fusarium wilt.
5. The Trichoderma QTYC44 strain provided by the invention is used for preventing and controlling plant soil-borne diseases and has no pollution to the environment.
Drawings
FIG. 1 shows the variation curve of Trichoderma QTYC44 chitinase activity with fermentation time
FIG. 2 the antagonistic culture effect of Trichoderma QTYC44 on three plant pathogenic bacteria
a. b and c are QTYC44 and experiments of confronting cucumber fusarium wilt bacteria, wheat gibberella and tobacco alternaria alternata respectively;
d. e and f are respectively cucumber fusarium wilt bacteria, wheat gibberella and tobacco brown spot bacteria control groups
FIG. 3 shows the control effect of Trichoderma QTYC44 on cucumber fusarium oxysporum
Detailed Description
1. The preparation and enzyme activity determination method of chitinase of trichoderma QTYC44 comprises the following steps:
(1) and (3) activation: inoculating Trichoderma QTYC44 with preservation number of CCTCC M2014267 on a PDA solid culture medium, and performing activated culture at 28 ℃ for 4-7 days to obtain an activated strain;
(2) inoculating the activated strain prepared in the step (1) into a MYG liquid culture medium for fermentation, and culturing for 3-4d at the temperature of 28 ℃ and under the condition of pH 7.0 to prepare a seed solution;
(3) inoculating the seed solution prepared in the step (2) into a chitinase induction culture medium for fermentation, and culturing for 6-9 days at the temperature of 28 ℃ and under the condition of pH 7.0 to prepare a fermentation liquid;
(4) and (4) centrifuging the fermentation liquor prepared in the step (3) for 10min at 8000r/min, and taking supernatant liquid as crude enzyme liquid every day at 3-9 d.
0.5mL of the crude enzyme solution, 1.5mL of 0.5mM pH7.8 phosphate buffer solution, and 1.0mL of colloidal chitin solution were added to the test tube, and the mixture was shaken and mixed, and then reacted in a 40 ℃ water bath for 90 min. Then centrifuging at 8000r/min for 10min, taking 3mL of supernatant, adding 1.5mL of DNS reagent, shaking, mixing uniformly, and boiling water bath for 10 min. After cooling, distilled water was added to each tube to 10mL, and absorbance was measured at 540nm with a spectrophotometer. And the control is that the enzyme is inactivated by boiling water bath for 10min before the enzyme solution to be detected is added into the chitin colloid. Each sample was replicated three times and the mean was taken and recorded as OD515nmThe absorbance of the control was then subtracted and the difference recorded as Δ a515 nm. And calculating the chitinase activity of the crude enzyme solution according to the light absorption value difference of the sample.
Chitinase activity: one chitinase enzyme activity unit (1U) is defined as the amount of enzyme required to catalyze the reaction of colloidal chitin at 40 ℃ and pH7.8 to release 1. mu. mol NAG per minute.
The variation of chitinase activity of Trichoderma QTYC44 with fermentation time is shown in FIG. 1. Chitinase activity increased from 3d to 5d, with 5d reaching a maximum of 0.1429U/mL.
2. In-vitro antagonistic activity of trichoderma QTYC44 on phytopathogen
The in vitro antagonistic activity test of Trichoderma sp.QTYC44 on plant pathogenic fungi is carried out by adopting an agar method. Inoculating Trichoderma QTYC44 to plant pathogenic fungi to PDA culture medium for activation culture for 3-4 d. A punch with diameter of 6mm is used to take Trichoderma QTYC44 and bacterial cake of plant pathogenic fungi, the bacterial cake is symmetrically attached to two sides of PDA plate at a distance of 4cm, and the blank control group is used for inoculating only plant pathogenic fungi. Each group is repeated three times, cultured for 5 days at 28 ℃, diameter data of plant pathogenic bacteria are measured, and the bacteriostasis rate is calculated.
The results of the experiments on the confronting of Trichoderma sp.QTYC44 and plant pathogenic fungi are shown in Table 1, the confronting effect is shown in figure 2, the Trichoderma sp.QTYC44 shows good inhibition effects on cucumber fusarium wilt bacteria, wheat fusarium graminearum bacteria and tobacco alternaria alternata bacteria, and the inhibition rates are 80.3%, 94.3% and 87.6% respectively.
TABLE 1 radius of inhibition (mm) of Trichoderma sp. QTYC44 against phytopathogenic fungi
3. Prevention and treatment effect of trichoderma QTYC44 on fusarium oxysporum
The living activity of Trichoderma sp.QTYC44 on cucumber fusarium oxysporum is tested by root irrigation. Taking healthy cucumber seeds (Jinyan No. four, Jiuquanchang agricultural development Co., Ltd.) for surface disinfection: sequentially soaking in 2% NaClO solution for 3min, soaking in 75% alcohol for 2min, and washing with sterile water for 3 times. Then spreading on moist sterile filter paper for accelerating germination, selecting cucumber seeds with the same growth vigor and sprouting after 2d, transplanting into a culture bowl, and culturing in an illumination incubator. The light incubator is set at 28 ℃, the light is set for 10 hours, the dark is set for 14 hours, and the water is poured once every two days.
When the cucumber seedlings grow to the two-leaf one-heart stage, 20mL 10mL is used6The roots of cucumber seedlings are irrigated with cfu/mL cucumber fusarium wilt germ spore suspension, and after 2 days, the roots are irrigated with 20mL 107cfu/mL of Trichoderma QTYC44 spore suspension, and the blank control group was root-irrigated with sterile water. 2 seedlings per pot, 12 pots per treatment as replicates. And (4) regularly observing the growth and disease incidence conditions of the cucumber seedlings, and performing statistical analysis on the biological control effect after 20 days. The cucumber fusarium wilt classification criteria are shown in table 2. And calculating disease index and prevention effect according to the statistical result and the following formula.
TABLE 2 grading Standard of cucumber fusarium wilt
The result of the pot culture test of the Trichoderma QTYC44 spore liquid on cucumber fusarium wilt is shown in figure 3, through 20d pot culture survey, the Trichoderma sp.QTYC44 spore liquid has obvious effect on preventing and treating cucumber fusarium wilt, and the disease index of a control group is 64.58%; the disease index of the experimental group is 17.71 percent, and the control effect of the Trichoderma sp.QTYC44 spore liquid on cucumber fusarium wilt is as high as 72.58 percent.
Claims (1)
1. Trichoderma (Trichoderma sp.) The application of QTYC44 in inhibiting plant pathogenic bacteria is disclosed, wherein the preservation number of the Trichoderma QTYC44 is CCTCC M2014267, and the pathogenic bacteria are cucumber fusarium wilt, wheat gibberella and tobacco brown spot.
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| US5260213A (en) * | 1987-04-03 | 1993-11-09 | Cornell Research Foundation, Inc. | Fused biocontrol agents |
| ATE271602T1 (en) * | 2000-05-02 | 2004-08-15 | Young-Ryun Chung | MICROBIAL PESTICIDE ACTIVE AGAINST PLANT FUNGAL PATHOGENS AND METHOD FOR PRODUCING THE SAME |
| CN102864081A (en) * | 2012-08-07 | 2013-01-09 | 上海交通大学 | Trichoderma strain for antagonizing cucumber fusarium wilt disease efficiently and application thereof |
| CN104099252B (en) * | 2014-07-09 | 2016-04-13 | 浙江师范大学 | Dragonfly larva enteron aisle cellulose degradation fungi and application thereof |
| CN105331544A (en) * | 2015-11-03 | 2016-02-17 | 红河学院 | Multifunctional trichoderma strain LY3 and preparation method for bacterium agent of multifunctional trichoderma strain |
| CN107043711B (en) * | 2017-04-26 | 2020-08-21 | 山东省农业科学院农产品研究所 | Trichoderma aureoviride T-321010 strain and culture method and application thereof |
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