CN112266879B - Trichoderma harzianum SQ-18 strain and application thereof in preventing and treating turfgrass diseases - Google Patents

Trichoderma harzianum SQ-18 strain and application thereof in preventing and treating turfgrass diseases Download PDF

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CN112266879B
CN112266879B CN202011011255.XA CN202011011255A CN112266879B CN 112266879 B CN112266879 B CN 112266879B CN 202011011255 A CN202011011255 A CN 202011011255A CN 112266879 B CN112266879 B CN 112266879B
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trichoderma harzianum
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章武
杨锦玉
卢翔
林金梅
牛学礼
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Abstract

The invention discloses a Trichoderma harzianum SQ-18 strain and application thereof in preventing and treating turfgrass diseases. The invention provides a Trichoderma harzianum SQ-18 strain which is preserved in Guangdong province microorganism strain preservation center in 2018, 7 and 26 months, wherein the preservation number is GDMCC No:60422. the Trichoderma harzianum SQ-18 strain has obvious inhibition effect on rhizoctonia zeae, phanerochaete licheniformis, botrytis cinerea, colletotrichum gloeosporioides and rhizoctonia solani, and the secondary metabolite of the Trichoderma harzianum SQ-18 strain has strong antagonistic effect on alternaria turfgrass, sporotrichum globosum and coriolus licheniformis; the bacterium liquid coarse substance of the Trichoderma harzianum SQ-18 strain has high inhibition rate on the lawn grass coin spot germ and the eupatorium rosenbergii; therefore, the Trichoderma harzianum SQ-18 strain, or the secondary metabolite thereof, or the bacterial liquid crude product thereof has wide application prospects in preventing and treating diseases caused by turfgrass pathogenic bacteria or preparing turfgrass disease prevention and treatment preparations.

Description

Trichoderma harzianum SQ-18 strain and application thereof in preventing and treating turfgrass diseases
Technical Field
The invention belongs to the technical field of biological control. More particularly, relates to a Trichoderma harzianum SQ-18 strain and application thereof in preventing and treating turfgrass diseases.
Background
In recent years, the diversity and pathogenicity of plant pathogenic bacteria have been increasing, and the causes of the increase are not only related to extreme weather caused by climate change, but also related to the long-term wide use of a large amount of pesticides such as herbicides and bactericides, and the like, which seriously affect the growth, yield, environment and ecological safety of plants. Therefore, the utilization of antagonistic microorganisms for preventing and treating the occurrence and harm of plant diseases at home and abroad has become one of the key points and hotspots of the current research.
Trichoderma (Trichoderma spp.) is a fungus of the kingdom fungi, deuteromycota, class Hyphomycetes, order Hyphomycetales, family Conidiobolus, genus Trichoderma. It is an antagonistic microorganism which is ubiquitous in nature and rich in resources, and has antagonistic action on plant pathogenic fungi and bacteria by producing bioactive substances such as antibiotics and cell wall decomposition enzymes, or by means of nutrient competition, micro parasitism, induction of plant resistance and the like, thereby achieving the effect of preventing and treating plant diseases. Since Weidling discovered that trichoderma has antagonistic action on phytopathogens for the first time in the 30s of the 20 th century, trichoderma is gradually and widely applied to biological control of phytopathogens as a biological control factor; for example, trichoderma harzianum is used for controlling sclerotinia rot of soybean, trichoderma atroviride is used for controlling Rhizopus solani, and the like. Various trichoderma biocontrol products have been registered, such as T22 (trichoderma harzianum) in the united states and T39 (trichoderma harzianum) in israel, among others. Therefore, the trichoderma has great application prospect in the fields of biological control of plant diseases, environmental protection and the like.
The lawn as an important green area plays irreplaceable important roles in beautifying the environment, purifying the air, maintaining water and soil, and providing outdoor activities and sports, but the lawn grass diseases are important factors influencing various functions and sustainable development of the lawn, and the lawn disease management and control measures which are mainly based on chemical control methods at present are one of main factors limiting the healthy development of the lawn disease management and control measures. Biological control of lawn grass diseases is a research hotspot of the current lawn management technology, and the bacterial treatment has the advantages of rich resources, strong selectivity, no residue, no pollution, low cost, prevention and treatment, and long-acting effect on keeping ecological balance; however, studies on the prevention and control of lawn diseases by trichoderma harzianum are relatively few in domestic and foreign researches, and only applications of trichoderma harzianum in the prevention and control of lawn fusarium wilt (Chinese patent with publication number CN103952320A and publication number 2014, 7 and 30) and lawn brown spot (Chinese patent with publication number CN103952321A and publication number 2014, 7 and 30) are reported. Therefore, the method continues to excavate and explore trichoderma resources with biocontrol effect on turfgrass pathogenic bacteria, and has important significance for preventing and treating turfgrass diseases.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing turfgrass disease control method and provides a Trichoderma harzianum SQ-18 strain and application thereof in turfgrass disease control.
The invention aims to provide a Trichoderma harzianum SQ-18 strain.
The invention also aims to provide application of the Trichoderma harzianum SQ-18 strain in preventing and treating diseases caused by pathogenic bacteria of turfgrass or preparing turfgrass disease prevention and treatment preparations.
The invention also aims to provide application of the secondary metabolite of the Trichoderma harzianum SQ-18 strain in preventing and treating diseases caused by turfgrass pathogenic bacteria or preparing turfgrass disease prevention and treatment preparations.
The invention also aims to provide the application of the bacterial liquid coarse substance of the Trichoderma harzianum SQ-18 strain in preventing and treating diseases caused by turfgrass pathogenic bacteria or preparing turfgrass disease prevention and treatment preparations.
The invention also aims to provide a turfgrass disease control preparation.
The above purpose of the invention is realized by the following technical scheme:
the invention provides a Trichoderma harzianum SQ-18 strain which is preserved in Guangdong province microorganism strain preservation center in 2018, 7 and 26 months, wherein the preservation number is GDMCC No:60422, the preservation address is Guangzhou city's Xielizhonglu No. 100 large yard No. 59 building No. 5 building.
The research of the invention finds that the Trichoderma harzianum SQ-18 strain has the inhibition rate of 100% to the rhizoctonia zeae and the phanerochaete licheniformis, the inhibition rate of more than 80% to the botrytis cinerea, the carpet grass anthracnose and the rhizoctonia solani, and the average inhibition rate of 72.8% to the pathogenic bacteria to be tested; the secondary metabolite of the Trichoderma harzianum SQ-18 strain has strong antagonistic effect on Sphaerotheca fuliginea, sporotrichum globosum and Phanerochaete licheniformis; the bacterial liquid crude substance of the Trichoderma harzianum SQ-18 strain has relatively high inhibition rate on lawn grass coin spot pathogen and Helminthosporium pernicifolium; therefore, the following applications should be within the scope of the present invention:
the Trichoderma harzianum SQ-18 strain is applied to the prevention and treatment of diseases caused by pathogenic bacteria of turfgrass or the preparation of turfgrass disease prevention and treatment preparations.
Preferably, the turfgrass pathogenic bacteria are any one or more of rhizoctonia zeae, phanerochaete licheniformis, botrytis cinerea, sorangium carpeting or rhizoctonia solani.
The secondary metabolite of the Trichoderma harzianum SQ-18 strain is applied to the prevention and treatment of diseases caused by pathogenic bacteria of turfgrass or the preparation of turfgrass disease prevention and treatment preparations.
Preferably, the turfgrass pathogenic bacteria is any one or more of turfgrass alternaria leaf spot pathogen, nigrospora sphaerica or coriolus licheniformis.
The bacterial liquid crude body of the Trichoderma harzianum SQ-18 strain is applied to the prevention and treatment of diseases caused by lawn grass pathogenic bacteria or the preparation of a lawn grass disease prevention and treatment preparation.
Preferably, the turfgrass pathogenic bacteria are turfgrass spot pathogen or helminthosporium pernyi.
The invention also provides a turfgrass disease control preparation, which comprises the Trichoderma harzianum SQ-18 strain, or a secondary metabolite thereof, or a bacterial liquid coarse substance thereof.
Preferably, the turfgrass disease is a disease caused by rhizoctonia solani, phanerochaete licheniformis, botrytis cinerea, colletotrichum carpeting, rhizoctonia solani, alternaria turfgrass, alternaria sphaerica or helminthosporium pergoloides.
The invention has the following beneficial effects:
the invention provides a Trichoderma harzianum SQ-18 strain and application thereof in preventing and treating turfgrass diseases. The invention discovers that Trichoderma harzianum SQ-18 strain can invade the growth space of lawn grass pathogenic bacteria by generating bacteriostatic zones, cover and go deep into the pathogenic bacteria and mutually twine with the pathogenic bacteria to enable the hypha of the pathogenic bacteria to become thin, contract and even break, or directly penetrate into the pathogenic bacteria hypha to absorb nutrition to enable hypha cells to be dissolved; the Trichoderma harzianum SQ-18 strain has an inhibition rate of 100% to rhizoctonia zeae and phanerochaete licheniformis, an inhibition rate of more than 80% to Botrytis cinerea, anthragma carpesii and Rhizoctonia solani, and an average inhibition rate of 72.8% to pathogenic bacteria to be tested; the secondary metabolite of the Trichoderma harzianum SQ-18 strain has strong antagonistic effect on Sphaerotheca fuliginea, sporotrichum globosum and Phanerochaete licheniformis; the bacterial liquid coarse substance of the Trichoderma harzianum SQ-18 strain has relatively high inhibition rate on the lawn spodoptera fruticosa and the euonymus platyphylla; therefore, the Trichoderma harzianum SQ-18 strain, or the secondary metabolite thereof, or the bacterial liquid crude product thereof has wide application prospects in preventing and treating diseases caused by turfgrass pathogenic bacteria or preparing turfgrass disease prevention and treatment preparations.
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FIG. 1 is a graph showing the antagonistic effect of 4 Trichoderma strains on 14 turf grass pathogenic bacteria; wherein, the picture (A) shows the growth conditions of 4 trichoderma strains and 14 lawn grass pathogenic bacteria plates during the opposite culture of 5 d; (B) The inhibition rates of 4 trichoderma strains on 14 lawn grass pathogenic bacteria during opposite culture at the 5 th day are shown in the figure, the numbers in the figure represent standard differences, and different lower case letters in the same column represent that the differences among different pathogenic bacteria under the same trichoderma strain are obvious (P < 0.05); (C) The overall inhibition of 14 turf grass pathogens by 4 trichoderma species is shown as the mean.
FIG. 2 is a morphogram of SQ-18 strain; wherein, the picture (A) is a morphogram of a spore-forming cell; (B) the picture is the morphogram of the molecular spore; the scale in the figure is 10 μm.
FIG. 3 is a graph of the mode of action of Trichoderma harzianum SQ-18 strain on lawn grass pathogenic bacteria hyphae; wherein, the picture (A) is a state picture that the hypha of Trichoderma harzianum SQ-18 strain is wound on the pathogenic bacteria mycelium; (B) FIG. 1 shows the state of pathogenic bacteria hyphae thinning and shrinking caused by Trichoderma harzianum SQ-18 strain; (C) FIG. is a diagram showing the state of Trichoderma harzianum SQ-18 strain causing hypha rupture of a pathogenic bacterium; (D) FIG. is a diagram showing the state of penetration of Trichoderma harzianum SQ-18 strain into the mycelia of pathogenic bacteria; (E) FIG. is a diagram showing a state in which Trichoderma harzianum SQ-18 strain is parasitized on the surface of pathogenic bacteria hyphae; (F) FIG. is a diagram showing the state of degradation of pathogenic bacteria hyphal cells by Trichoderma harzianum SQ-18 strain; the scale in the figure is 20 μm.
FIG. 4 is a graph showing the antagonistic effect of the secondary metabolites of Trichoderma harzianum SQ-18 strain and crude extracts thereof on turf grass pathogenic bacteria; wherein, the picture (A) is a picture of the antagonistic effect of secondary metabolites of Trichoderma harzianum SQ-18 strain on lawn grass pathogenic bacteria; (B) The antagonistic effect of the crude extract of the bacterial liquid of Trichoderma harzianum SQ-18 strain on pathogenic bacteria of turfgrass is shown.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 screening and identification of Trichoderma harzianum SQ-18 Strain
1. Experimental methods
The test 4 Trichoderma strains are numbered as follows: SQ-18 (Hainan, mitsui, shenquan International Golf course), SQ-1Q-15 (Hainan, mitsui, shenquan International Golf course), BD-1F-1 (Hainan, wanning, shenzhou Seandao Golf course), BD-2Q-12 (Hainan, wanning, shenzhou Seandao Golf course), and the information of the pathogenic bacteria of the lawn grass to be tested is shown in Table 1.
TABLE 1 information on pathogenic bacteria of turfgrass to be tested
Figure BDA0002697630980000041
Figure BDA0002697630980000051
(1) Bacterial activation
Respectively inoculating 4 trichoderma strains and 14 turfgrass pathogenic bacteria which are stored on the inclined plane at the low temperature of 4 ℃ to a PDA culture medium for activation, and placing the activated bacteria in a constant-temperature incubator at the temperature of 25 ℃ for culture for 5 days for later use.
(2) Determination of antibacterial activity by confrontation culture method
Marking specific positions of trichoderma and pathogenic bacteria for opposite culture on the back surface of a PDA culture medium plate respectively by using a marking pen (the trichoderma and the pathogenic bacteria are symmetrical by the central line of a culture dish and are spaced by 3 cm), then respectively punching and sampling 4 well-grown trichoderma and 14 kinds of pathogenic bacteria by using a sterilization puncher (d =5 mm) under an aseptic condition, picking fungus cakes by using an inoculating needle and respectively inoculating the fungus cakes on the surface of the PDA culture medium with the marked positions for opposite culture, and inoculating trichoderma and the pathogenic bacteria on each plate. The control is only inoculated with the pathogenic bacteria cake, and each confrontation experiment and the control are respectively provided with 4 times of repetition. Culturing in 25 deg.C constant temperature incubator, observing and recording opposite growth conditions of Trichoderma and pathogenic bacteria every day, measuring radius of colony of each treatment group and control group by "cross method" at 3d, taking pictures, measuring for 1 time every day, continuously measuring for 4d, and calculating growth inhibition rate according to the following growth inhibition rate formula.
Figure BDA0002697630980000052
Wherein GI is growth inhibition (%) and d 0 Control colony radius, d t To address colony radius.
(3) Identification of SQ-18 Strain
Morphological identification: after the SQ-18 strain is activated, the strain is cultured for 7d at the temperature of 25 ℃, the growth speed, colony color, surface characteristics and the like of the strain are observed, and morphological characteristics of the strain such as hypha, spore-forming cells, spores and the like are observed under a microscope.
Molecular biological identification: activating SQ-18 strain, culturing at 25 deg.C for 1 week, scraping hypha with sterile glass slide, grinding, extracting pathogenic bacteria genome DNA with CTAB method, and storing at-20 deg.C in refrigerator. PCR amplification was carried out using rDNA-ITS region universal primers ITS-1 (5-. PCR amplification System: 10 XPCR Buffer 5U L, dNTP mix 4U L, 20U mol/L primer each 1U L, 5U/L Taq enzyme 0.25U L, template DNA 1U L, adding sterile double distilled water to 50U L. And (3) PCR reaction conditions: preheating at 94 deg.C for 4min; 30s at 94 deg.C, 30s at 62 deg.C, and 1min at 72 deg.C for 34 cycles; extension at 72 ℃ for 5min. All PCR products were electrophoresed in 1.0% agarose gels, stained with ethidium bromide, and examined with a gel imager. The PCR product was sent to Biotechnology engineering (Shanghai) Co., ltd for DNA sequencing. The determined ITS sequences are submitted to NCBI nucleic acid database to obtain GenBank accession number, and meanwhile, the Blastn software in GenBank database is used for comparison and homology analysis. And finally, identifying the trichoderma by combining the analysis result with morphological characteristics.
2. Results of the experiment
Antagonistic effects of 4 trichoderma strains on 14 turf grass pathogenic bacteria are shown in fig. 1, wherein (a) shows the growth conditions of the 4 trichoderma strains and the 14 turf grass pathogenic bacteria plates when the plates are oppositely cultured for 5d, (B) shows the inhibition rates of the 4 trichoderma strains on the 14 turf grass pathogenic bacteria when the plates are oppositely cultured for 5d, and (C) shows the total inhibition rates of the 4 trichoderma strains on the 14 turf grass pathogenic bacteria; as can be seen, 4 strains of Trichoderma hyphae begin to contact with pathogenic bacteria hyphae during the opposite culture for 2 days; at 5d, 4 Trichoderma strains had different degrees of antagonism against the 14 pathogenic fungi tested, compared to the control; among them, the SQ-18 strain had the most significant antagonistic effect. The 4 trichoderma strains have the strongest inhibition effect on the phanerochaete Licheniformis (LF), and the inhibition effect is as high as 100%; secondly, the strain has strong inhibiting effect on Rhizoctonia Zeae (RZ), wherein the inhibiting rate of the SQ-18 strain and the strain BD-1F-1 on the Rhizoctonia zeae is as high as 100%.
In addition, in addition to the phanerochaete licheniformis and rhizoctonia solani, 4 trichoderma strains have obvious inhibiting effect on the other 12 tested pathogenic bacteria, and the SQ-18 strain has the strongest inhibiting effect on rhizoctonia solani (R.solani, RS), colletotrichum gloeosporioides (CH), paspalum vaginatum (MP), nigrospora Sphaerica (NS) and verticillium pernicicola (BP), and the inhibiting rates are respectively 95.5%, 88.5%, 78.0%, 61.6% and 59.0%; the SQ-18 strain Q-15 has the strongest inhibition effect on the lawn grass spot disease pathogenic bacteria (SH), and the inhibition rate is 79.4%; the strain BD-1F-1 has the strongest inhibition effect on bacterial blotch (Curvularia malina, CM), phoma graminearum (Phoma herbarum, PH) and Curvularia lunata (Cu.lunata, CL), and the inhibition rates are 70.0%,55.4% and 44.0% respectively; the strain BD-2Q-12 showed the strongest inhibitory effect on Botrytis Cinerea (BC), graves Gramineae (GG) and Fusarium Equiseti (FE), with the inhibitory rates of 92.15%,81.9% and 69.2%, respectively.
The above results show that: the SQ-18 strain has the strongest average inhibiting effect on 14 pathogenic bacteria to be tested, the inhibiting rate on the rhizoctonia zeae and the phanerochaete licheniformis is up to 100 percent, the inhibiting rate on the botrytis cinerea, the sorghumus carpesii and the rhizoctonia solani is more than 80 percent, the average inhibiting rate on the pathogenic bacteria to be tested is up to 72.8 percent, the mean value dispersion is moderate, and the distribution density is relatively uniform.
The morphogram of SQ-18 strain is shown in FIG. 2, wherein (A) is the morphogram of spore-forming cells; (B) the picture is the morphogram of the molecular spore; as can be seen, after the SQ-18 strain was activated on PDA medium, aerial hyphae grew rapidly. The colony is white and felty in the initial stage, a large number of spore clusters are formed in the later stage and become light green, the spore clusters are densely paved on a culture medium plate, and the back surface of the colony is yellow green or golden yellow. Conidiophores are arranged in a typical pyramid structure, primary branches are distributed at intervals, and a large number of secondary branches are paired. The bottle stem cells are single or 2-5 rounds, and the shape of the flask is changed into the shape of an ampoule. The conidium is nearly spherical or ellipsoidal, the surface is smooth and light green, the conidium is dark green after aging, and the SQ-18 strain is preliminarily identified as Trichoderma harzianum (Trichoderma harzianum) according to the morphological characteristics.
The ITS fragment obtained by sequencing the SQ-18 strain has the length of 595bp, the sequence is proved to have high similarity with a plurality of Trichoderma harzianum (Trichoderma harzianum) sequences (MK 738145, MN658589 and MN 602615) through BLASTn comparison analysis of a GenBank website, the sequence is further identified as Trichoderma harzianum (Trichoderma harzianum) through combining morphological characteristics and is named as Trichoderma harzianum (Trichoderma harzianum) SQ-18 strain, and the strain is preserved in Guangdong province microbial strain collection center in 7 and 26 months in 2018, and the preservation number is GDMCC No:60422, the preservation address is No. 59 building 5 of No. 100 Dazhong Jie-Lu-100, guangzhou city.
Example 2 measurement of bacteriostatic Effect of Trichoderma harzianum SQ-18 Strain
1. Experimental method
(1) Determination of antagonistic grades
Trichoderma colonies completely occupy the whole culture dish, namely the occupied area =100%, and the grade is I; 3/4< trichoderma colonies occupy the area of the culture dish to be less than 1, and the grade is II; 2/3< trichoderma colonies occupy the area of the culture dish to be less than 3/4, and the grade is III; 1/3< trichoderma colonies occupies less than 2/3 of the area of the culture dish, and the grade is IV; 0< trichoderma colonies occupy <1/3 of the area of the culture dish, and the grade is V; the pathogenic bacteria colony occupies 100% of the culture dish area and is graded VI.
(2) Microscopic observation of bacteriostatic action mechanism
Under the aseptic condition, a pipette is used for sucking 1mL of aseptic PDA culture medium, and the aseptic PDA culture medium is uniformly smeared on a glass slide to prepare a layer of PDA culture medium film. After the culture medium on the glass slide is solidified, respectively inoculating Trichoderma harzianum SQ-18 strain and 14 lawn grass pathogenic bacteria at two ends of the glass slide, wherein the distance between the Trichoderma and the pathogenic bacteria is 3cm, then placing the glass slide in a sterile culture dish, sealing a film, and culturing in a constant-temperature incubator at 25 ℃, wherein the treatment is repeated for 3 times. After 3d of culture, when the two colonies are mutually connected, the glass slide is placed under an optical microscope to observe the morphological characteristics of the colonies after the interaction of trichoderma and 14 pathogenic bacteria, and the colonies are recorded and photographed.
(3) Determination of bacteriostatic activity by cellophane method
Cutting cellophane into disks with diameter of 9cm, sterilizing with high pressure steam, and attaching to the center of PDA culture medium (diameter =9 cm). The Trichoderma harzianum SQ-18 strain cake is inoculated to the center of a culture medium containing glass paper and cultured under the dark condition at the temperature of 25 ℃. When the trichoderma colonies are close to the edges of the cellophane, the cellophane and the whole colonies are removed, and the center of the culture dish is inoculated with the fungus cakes of various pathogenic bacteria. The control was made by inoculating the pathogen into untreated PDA medium. Each treatment and control set up was 3 replicates. And observing the growth condition of each pathogenic bacterium every day, measuring the diameter of the pathogenic bacterium on the third day, taking a picture, and calculating the bacteriostatic effect according to a growth inhibition rate formula.
(4) Determination of antibacterial activity by trichoderma liquid crude extract
The trichoderma cake was picked up with a sterilized punch (d =5 mm) on an activated PDA plate, picked up with an inoculating needle and inoculated in a 500mL flask containing 150mL of Potato Dextrose Broth (PDB), inoculated in 10 bottles in total, shake-cultured at 28 ℃ for 5d on a shaker at 150r/min, filtered off with sterile absorbent cotton, and filtered with sterile filter paper. Adding equal volume of ethyl acetate into the obtained bacterial liquid, soaking for 24h, extracting for 3 times in each bottle, combining the extract liquid, and concentrating at 45 ℃ by a rotary evaporator to obtain the crude extract of the fermentation liquid. The crude extract was dissolved in acetone to prepare a solution (10 mg. Multidot.mL) -1 ) 10 mul of crude extract solution was spread evenly on a petri dish containing PDA medium using a pipette, then a cake of pathogenic bacteria was inoculated in the center of the petri dish, using the same volume of acetone solution as a control, and incubated in a constant temperature incubator at 25 ℃ in the dark, with each treatment repeated 3 times. And recording the growth condition of hypha of the pathogenic bacteria every day, measuring the diameter of the pathogenic bacteria on the third day, taking a picture, and calculating the bacteriostatic effect according to a growth inhibition rate formula.
2. Results of the experiment
(1) Observation and analysis of antagonistic culture of Trichoderma harzianum SQ-18 strain on 14 lawn grass pathogenic bacteria
The antagonistic effect and the grade of the Trichoderma harzianum SQ-18 strain on the growth of 14 pathogenic bacteria are shown in Table 2, and the Trichoderma harzianum SQ-18 strain has obvious inhibition effect on the growth of 14 turfgrass pathogenic bacteria to be tested, but the Trichoderma harzianum SQ-18 strain has larger difference on different pathogenic bacteria antagonistic grades; wherein, the antagonism grades of the Trichoderma harzianum SQ-18 strain to the Phanerochaete licheniformis and the rhizoctonia zeae are I grades, namely the antagonism is the strongest; the antagonistic grade of the alternaria alternate is II grade on the alternaria alternate, the helminthosporium pervirens, the rhizoctonia solani, the anthracnose of carpet grass, the botrytis cinerea, the gaeumannomyces graminis and the alternaria alternata; the antagonistic grade of the fusarium equiseti and sclerotinia sclerotiorum is grade III; the antagonism grades for curvularia lunata, nigrospora sphaerica and phoma herbarum are IV grades.
TABLE 2 antagonistic effect and grade of Trichoderma harzianum SQ-18 strain on the growth of 14 pathogenic bacteria
Figure BDA0002697630980000091
Figure BDA0002697630980000101
(2) Microscopic observation of trichoderma harzianum SQ-18 strain on bacteriostatic action mechanism of lawn grass pathogenic bacteria
The action mode of Trichoderma harzianum SQ-18 strain on lawn grass pathogenic bacteria mycelium is shown in FIG. 3, wherein (A) is a state diagram of Trichoderma harzianum SQ-18 strain mycelium wound on the pathogenic bacteria mycelium; (B) FIG. 1 shows the state of pathogenic bacteria hyphae thinning and shrinking caused by Trichoderma harzianum SQ-18 strain; (C) FIG. is a diagram showing the state of Trichoderma harzianum SQ-18 strain causing hypha rupture of a pathogenic bacterium; (D) FIG. is a diagram showing the state of penetration of Trichoderma harzianum SQ-18 strain into the mycelia of pathogenic bacteria; (E) The figure is a state diagram of the trichoderma harzianum SQ-18 strain parasitizing on the surface of the hypha of the pathogenic bacteria; (F) FIG. is a diagram showing the state of degradation of pathogenic bacteria hyphal cells by Trichoderma harzianum SQ-18 strain; it can be seen that the inhibition action modes of the Trichoderma harzianum SQ-18 strain on 14 pathogenic bacteria are similar, mainly after two kinds of fungi are mutually contacted, the Trichoderma harzianum SQ-18 strain quickly invades a pathogenic bacteria growth space, and then the mycelium of the Trichoderma harzianum SQ-18 strain is wound on the pathogenic bacteria mycelium and staggered with the pathogenic bacteria, so that the pathogenic bacteria mycelium becomes thin and is constricted; even leading to the rupture of the hyphae of the pathogenic bacteria, or penetrating into the hyphae of the pathogenic bacteria or parasitizing on the surface of the hyphae when the pathogenic bacteria are serious, and concentrating the protoplasm of the hyphae cells of the pathogenic bacteria until the hyphae cells of the pathogenic bacteria are degraded finally.
The above results show that: the Trichoderma harzianum SQ-18 strain can invade the growth space of the pathogenic bacteria of the lawn grass by generating bacteriostatic zones, cover and go deep into the pathogenic bacteria and mutually twine with the pathogenic bacteria to enable the hypha of the pathogenic bacteria to become thin, contract and even break, or directly penetrate into the pathogenic bacteria hypha to absorb nutrition to enable the hypha cells to be dissolved.
(3) Antagonistic effect of secondary metabolite of Trichoderma harzianum SQ-18 strain and crude extract of strain liquid thereof on pathogenic bacteria of turfgrass
The antagonistic effect of the secondary metabolite of Trichoderma harzianum SQ-18 strain and its crude extract on pathogenic bacteria of lawn grass is shown in FIG. 4, wherein (A) is antagonistic effect of the secondary metabolite of Trichoderma harzianum SQ-18 strain on pathogenic bacteria of lawn grass; as can be seen, compared with the control, the secondary metabolite of the Trichoderma harzianum SQ-18 strain has antagonistic effect on the growth of 13 pathogenic bacteria except Rhizoctonia solani, and the secondary metabolite generated by penetrating glass paper in the growth process of the Trichoderma harzianum SQ-18 strain has no antagonistic effect on the Rhizoctonia solani, has inhibitory effect on other 13 pathogenic bacteria in different degrees, and the inhibitory rate on the same pathogenic bacteria changes along with the culture time. After the pathogenic bacteria are cultured for 3 days, the Trichoderma harzianum SQ-18 strain has stronger antagonistic action on the pathogenic bacteria of the lawn grass spot disease and the Neurospora sphaericoides, and the relative inhibition rates of the Trichoderma harzianum SQ-18 strain are respectively up to 91.77% and 89.80% when the relative inhibition rates are the highest; when the culture reaches the 5d, the inhibition rates are 89.63 percent and 76.83 percent respectively; wherein the inhibition rate of the rhizoctonia zeae at 3d is 37.8%, and the inhibition rate is reduced to 0% at 4 th and 5 th days.
Further, the antagonistic effect of the crude extract of the fungal solution of Trichoderma harzianum SQ-18 strain against pathogenic bacteria of turf grass is shown in FIG. 3 (B), and it can be seen that, the crude extract of the bacterial liquid of the Trichoderma harzianum SQ-18 strain obtained by ethyl acetate extraction and rotary evaporator concentration and evaporation to dryness has no obvious inhibition effect on 14 turfgrass pathogenic bacteria, and the inhibition rate is below 50%; the inhibition effect on various pathogenic bacteria is different, wherein the inhibition rates on the lawn pennywort herb spot pathogen and the eupatorium rosenbergii are relatively high and are respectively 43.60 percent and 42.91 percent; the inhibition rate of curvularia lunata, phoma herbarum and nigrospora sphaerica is lower than 5 percent; while rhizoctonia solani and rhizoctonia solani have no inhibitory effect.
The above results show that: the Trichoderma harzianum SQ-18 strain can produce antagonistic compounds with inhibitory activity on pathogenic bacteria of turfgrass, wherein secondary metabolites (antagonistic substances) of the Trichoderma harzianum SQ-18 strain produced by a glassine method have strong antagonistic effects on alternaria turmerina, nigrospora sphaerica and coriolus licheniformis, and after 3d inoculation, the relative inhibition rates are respectively up to 91.77%, 89.80% and 79.59%; the bacterial liquid coarse substance of the Trichoderma harzianum SQ-18 strain has relatively high inhibition rate on alternaria turcica and helminthosporium pernicifluum, which is 43.60% and 42.91% respectively.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (5)

1. Trichoderma harzianum (f) (ii)Trichoderma harzianum) SQ-18 strain, wherein said strain has been deposited at 26.7.2018 with the culture Collection of microorganisms of Guangdong province under the accession number GDMCC No:60422.
2. the use of a trichoderma harzianum SQ-18 strain of claim 1 for controlling diseases caused by turfgrass pathogenic bacteria or for the preparation of a turfgrass disease control formulation, wherein said turfgrass pathogenic bacteria is any one or more of rhizoctonia solani, rhizoctonia licheniformis, botrytis cinerea, trichoderma carposum or rhizoctonia solani.
3. The use of a secondary metabolite of the trichoderma harzianum SQ-18 strain of claim 1 for controlling diseases caused by turfgrass pathogenic bacteria or for preparing a turfgrass disease control preparation, wherein the secondary metabolite is obtained by inoculating a cake of the trichoderma harzianum SQ-18 strain into the center of a medium containing glassine paper, culturing at 25 ℃ in the dark, removing the glassine paper and the whole colony when a trichoderma colony approaches the edge of the glassine paper, and producing the secondary metabolite through the glassine paper; the lawn grass pathogenic bacteria are any one or more of lawn grass marssonina, neurospora sphaerica or dermiformis licheniformis.
4. The use of the crude extract of a bacterial liquid of the trichoderma harzianum SQ-18 strain of claim 1 for controlling diseases caused by pathogenic bacteria of turfgrass or for preparing a preparation for controlling diseases of turfgrass, wherein the crude extract of the bacterial liquid is obtained by extraction with ethyl acetate, concentration by a rotary evaporator and evaporation to dryness; the lawn grass pathogenic bacteria are lawn grass alternaria cucumerina or helminthosporium perniciatum.
5. A turfgrass disease control preparation, characterized by, comprising Trichoderma harzianum SQ-18 strain as described in claim 1, or a secondary metabolite of Trichoderma harzianum SQ-18 strain as described in claim 3, or a crude extract of a bacterial broth of Trichoderma harzianum SQ-18 strain as described in claim 4.
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