CN114480143B - Trichoderma harzianum M6 for preventing and treating sclerotinia sclerotiorum of sunflower and application thereof - Google Patents

Trichoderma harzianum M6 for preventing and treating sclerotinia sclerotiorum of sunflower and application thereof Download PDF

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CN114480143B
CN114480143B CN202210240527.6A CN202210240527A CN114480143B CN 114480143 B CN114480143 B CN 114480143B CN 202210240527 A CN202210240527 A CN 202210240527A CN 114480143 B CN114480143 B CN 114480143B
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trichoderma harzianum
sunflower
sclerotium
strain
rot
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CN114480143A (en
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马立功
孟庆林
石凤梅
刘佳
李易初
苏保华
付晓晨
王军
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PLANT PROTECTION INSTITUTE OF HEILONGJIANG PROV AGRICULTURAL SCIENCES ACADEMY
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01N63/00Biocides, 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
    • A01N63/30Microbial fungi; Substances produced thereby or obtained therefrom
    • A01N63/38Trichoderma
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    • C12N1/00Microorganisms, 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
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    • Y02A40/00Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture

Abstract

The invention discloses trichoderma harzianum M6 for preventing and treating sclerotinia rot of sunflower and application thereof, wherein the preservation number of the trichoderma harzianum (Trichoderma harzianum) M6 in the China general microbiological culture collection center is CGMCC No.40041. The invention also provides a microbial inoculum, the active ingredient of which is Trichoderma harzianum (Trichoderma harzianum) M6 as claimed in claim 1. The Trichoderma harzianum M6 strain is a biocontrol fungus, can be prepared into biocontrol preparations for biological control of sunflower sclerotium disease, and has the characteristics of high efficiency, low toxicity, low residue, no pollution, difficult generation of drug resistance and the like. The Trichoderma harzianum M6 strain mycelium can wind sclerotinia sclerotiorum mycelium to grow, and the mycelium is inhibited from forming sclerotium. The trichoderma harzianum M6 strain conidium suspension has remarkable growth promoting effect on sunflower seedling growth and has good control effect on sunflower seedling rot and leaf rot sclerotium disease.

Description

Trichoderma harzianum M6 for preventing and treating sclerotinia sclerotiorum of sunflower and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to trichoderma harzianum M6 for preventing and treating sclerotinia sclerotiorum of sunflower and application thereof.
Background
Sclerotinia rot of sunflower, also known as white rot or rotten disk disease, is a disease of sunflower commonly occurring worldwide, and is particularly serious in places such as Heilongjiang, jilin, liaoning, inner Mongolia, shanxi and the like in China. In recent years, the epidemic outbreak of sclerotinia in the main production area of sunflowers in China causes serious yield loss, and even in some areas, the seed is stopped, so that the seeding area of the dominant crop is greatly reduced, and the healthy development of the sunflower planting industry is greatly influenced.
The pathogenic bacteria of sunflower sclerotinia are sclerotinia sclerotiorum (Sclerotinia sclerotiorum (Lib) de Bary.) belonging to the genus sclerotinia of the phylum ascomycotina, class Pantoea. The bacterial host range is very broad, up to 450 varieties, with sunflower being one of the most susceptible plants. The pathogenic bacteria have strong pathogenicity, can penetrate through the epidermis of a host plant to kill cells, further infect a plurality of parts such as the root, the stem, the leaf, the flower disc and the like of sunflower, even cause partial rot or death of the whole plant, and can cause great loss to the sunflower if improper management. Sclerotinia can occur from seedling stage to mature stage of sunflower, and causes root rot, stem rot, leaf rot, disk rot and other series of symptoms respectively. Wherein root rot symptoms are caused by hypha infection formed by sclerotium germination in field soil, and pan rot, stem rot and leaf rot symptoms are caused by ascospore infection formed by sclerotium germination. The Chinese is large in eastern and western span, the ecological climate types are various, and the symptoms of the sclerotinia sclerotiorum of the sunflower caused by the sclerotinia sclerotiorum infection are different. In the northeast, the eastern portion of inner mongolia and other wet and rainy regions, the disk rot type is the main part, while in the northwest (Xinjiang) arid regions, the root rot type is the main part.
Sclerotinia sclerotiorum is soil-dwelling bacteria, can survive and accumulate in soil for a long time, and 90% of the life history of sclerotium is in soil for a long time. Sclerotium can sleep on the surface of soil or between mixed seeds, has strong vitality, can survive for 6-8 years in drought soil and can survive for 3-5 years in moist soil. The number of sclerotium germination in the soil layer of 1-3 cm in the field is the largest, and the number of ascus discs generated by germination is very small below 4 cm. Sufficient soil humidity and proper temperature are the necessary conditions for sclerotium germination, and the sclerotium can germinate to form an ascus dish 20-30 days under the conditions of 12-22 ℃ and high humidity (60% -80%). Suitable aeration conditions are required for germination of sclerotium, which cannot germinate when immersed in water or buried in deeper soil (below 8 cm) or very compact soil; no light is required for sclerotium germination, but some scattered light is required for ascal formation. Blue and orange light can only form ascofacial, only under natural light or red, yellow and green light, and light intensity of more than 230lx and illumination of more than 8 hours are needed to form ascofacial.
At present, no effective method for preventing and treating sclerotinia rot of sunflower exists. As the natural world lacks good anti-source breeding materials, the disease-resistant breeding of sunflower sclerotinia disease has slow progress, and no disease-resistant variety or high-efficiency disease-resistant variety exists in production. The chemical prevention and treatment mainly uses the medicines such as carbendazim, secalin, dimethachlon and the like, but the sunflower is a high-stalk crop, the field application is very difficult, and the effect of the medicines on germs in soil is limited, so that the prevention and treatment effect is not ideal. In the aspect of agricultural control, a method which is economical and practical and has remarkable effect is also lacking. In addition, since seed tape and residual sclerotium in soil are the primary sources of initial infection in the next year of sunflower sclerotinia.
In summary, how to provide a beneficial microorganism for parasitizing or inhibiting the germination of sclerotium in soil to prevent and treat sunflower sclerotinia is a technical problem to be solved.
Disclosure of Invention
Therefore, the invention provides trichoderma harzianum M6 for preventing and treating sclerotinia sclerotiorum of sunflower and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the first aspect of the invention provides Trichoderma harzianum (Trichoderma harzianum) M6 with the preservation number of CGMCC No.40041 in China general microbiological culture Collection center.
In one embodiment of the invention, the 16S rDNA sequence of the Trichoderma harzianum (Trichoderma harzianum) M6 is shown in SEQ ID NO. 1.
The second aspect of the invention also provides a microbial inoculum, the active ingredient of which is the trichoderma harzianum (Trichoderma harzianum) M6.
In one embodiment of the invention, the trichoderma harzianum (Trichoderma harzianum) M6-wound sclerotinia mycelium grows, inhibiting mycelium from forming sclerotium;
the Trichoderma harzianum (Trichoderma harzianum) M6 conidium has parasitic peristalsis effect on sclerotium;
the trichoderma harzianum (Trichoderma harzianum) M6 has the effects of removing the sclerotium remained in the field soil and reducing the primary infection source of diseases;
the trichoderma harzianum (Trichoderma harzianum) M6 has the function of preventing and treating sunflower seedling rot and leaf rot sclerotium disease.
In one embodiment of the invention, the sclerotinia is a sclerotinia sclerotiorum SS-12 strain.
The third aspect of the invention also provides a product for preventing and treating sclerotinia sclerotiorum of sunflower or promoting the growth of sunflower or promoting the yield increase or antibiosis of sunflower, and the active ingredient of the product is trichoderma harzianum (Trichoderma harzianum) M6 or the microbial inoculum.
In one embodiment of the invention, the sclerotinia of sunflower is caused by sclerotinia sclerotiorum;
the antibacterial is an anti-sclerotinia sclerotiorum SS-12 strain;
the promotion of sunflower growth is manifested by an increase in sunflower plant height or an increase in sunflower biomass or an increase in sunflower seed yield.
The application of the trichoderma harzianum (Trichoderma harzianum) M6 or the biocontrol agent in preventing and treating sclerotinia of sunflowers or promoting the growth of sunflowers or promoting the yield increase or the antibiosis of sunflowers also belongs to the protection scope of the invention.
In one embodiment of the invention, the sclerotinia of sunflower is caused by sclerotinia sclerotiorum;
the antibacterial is an anti-sclerotinia SS-12 strain.
In one embodiment of the invention, the promotion of sunflower growth is manifested by an increase in sunflower plant height or an increase in sunflower biomass or an increase in sunflower seed yield.
The invention has the following advantages:
the Trichoderma harzianum M6 strain is a biocontrol fungus, can be prepared into biocontrol preparations for biological control of sunflower sclerotium disease, and has the characteristics of high efficiency, low toxicity, low residue, no pollution, difficult generation of drug resistance and the like.
The Trichoderma harzianum M6 strain hypha can wind sclerotinia hypha growth, the hypha is inhibited from forming sclerotium, and the Trichoderma harzianum M6 conidium has strong parasitic peristalsis effect on sclerotium, so that the method is suitable for removing sclerotium remained in field soil and reducing an initial infection source of diseases.
The trichoderma harzianum M6 strain spore suspension has remarkable growth promoting effect on sunflower seedling growth and has good control effect on sunflower seedling rot and leaf rot sclerotinia.
And (3) strain preservation: the trichoderma harzianum (Trichoderma harzianum) M6 is preserved in China general microbiological culture collection center (China center), and has the address of Hospital No. 3, north Chen West Lu No.1, the preservation number of CGMCC No.40041, and the preservation date of 2022, 1 month and 11 days.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.
The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the invention, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present invention, should fall within the ambit of the technical disclosure.
FIG. 1 is a morphological characteristic diagram of Trichoderma harzianum M6 provided by the embodiment of the present invention, wherein a is morphological characteristics of mycelium on PDA culture medium; b, hypha; conidiophores and conidiophores; d, tree branch conidiophore.
FIG. 2 is a phylogenetic tree of Trichoderma harzianum M6 and other Trichoderma strains provided by an embodiment of the present invention;
FIG. 3 is a photograph of sclerotium produced by sunflower sclerotium disease sclerotium disk strain SS-12 grown on potato-carrot medium according to an example of the invention;
FIG. 4 shows an indoor test of Trichoderma harzianum M6 parasitic sunflower sclerotium disease in which CK is a control treatment;
FIG. 5 shows the surface morphology of the sclerotium of the Trichoderma harzianum M6 parasitic sunflower sclerotium and browning and rot inside the sclerotium, provided by the embodiment of the invention;
FIG. 6 is a diagram showing a test of the opposite of Harz raspberry M6 to sclerotinia in the example of the present invention, wherein a is the front of the hypha opposite test; b, mycelium confrontation test back; c, winding M6 hyphae on sclerotinia sclerotiorum hyphae;
FIG. 7 is a graph showing that the Harz raspberry M6 provided in the examples of the present invention promotes sunflower seedling test at plant heights of 10 days, 20 days, and 30 days, respectively;
fig. 8 is an in vitro leaf blade test for preventing and treating sclerotinia sclerotiorum by using a suspension of harzia raspberry M6 conidium provided by the embodiment of the invention;
FIG. 9 is a diagram showing a test of a trichoderma harzianum M6 parasitic sunflower sclerotium bacteria nuclear potting according to an embodiment of the present invention, wherein CK is a control treatment;
fig. 10 shows a test of the effect of trichoderma harzianum M6 on controlling a sunflower seedling rot sclerotium disease pot according to the present invention, wherein CK is a control treatment.
Detailed Description
Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 isolation and identification of Trichoderma harzianum M6 Strain
1. Collection of sclerotium samples from sunflower sclerotium disease
Sclerotium was collected from sunflower sclerotium disease-onset fields in Heilongjiang, inner Mongolia, gansu and Xinjiang areas, 10 parts of sclerotium per area. The collecting method comprises randomly collecting sunflower field sclerotium type, stem rot type and root rot type sclerotium in different regions, packaging, marking, and placing in a sample bag for carrying to a laboratory.
2. Isolation and culture of strains
The sclerotium obtained from the field is placed in a germination box filled with sterile sand after surface sterilization, placed in a 22 ℃ incubator for culture, and after the green conidium grows out, the conidium is diluted and coated on a PDA plate to obtain a pure isolate of the single cell culture.
3. Identification of Trichoderma harzianum M6
3.1 morphological identification
Inoculating the separated and purified trichoderma strain on a culture medium containing PDA, culturing, picking hypha and conidium to prepare a slide after the culture dish is full, and observing the morphology of conidium peduncles and spores.
As shown in FIG. 1, the results showed that M6 was grown in a radial manner with the hyphae initially white and the fluffy shape on the PDA medium plate, the aerial hyphae developed, the colony appeared green, and pigment was accumulated in the medium with the extension of the culture time, and the back of the plate appeared yellow. The conidiophores are tree-shaped branches, the primary branches are right angles to the main shaft, and the conidiophores are spherical or oval.
3.2 molecular biological identification
Inoculating M6 strain onto PDA plate paved with a layer of sterilized cellophane, culturing at 25deg.C for 2d, collecting mycelium, and freezing at-80deg.C. The DNA of the M6 strain is extracted from a fungus genome by adopting a CTAB method, an ITS sequence of the DNA is amplified by PCR to obtain an amplified product, the amplified product is directly subjected to bidirectional sequencing after being purified, and the amplified ITS gene sequence is submitted to BLAST program analysis on NCBI and identified as Trichoderma harzianum (Trichoderma harzianum) which is named as Harz raspberry (Trichoderma harzianum) M6.
The PCR amplification primer sequences are ITS1 primer and ITS4 primer, the nucleotide sequence of the ITS1 primer is 5'-TCCGTAGGTGAACCTGCGG-3', ITS primer, and the nucleotide sequence of the ITS4 primer is 5'-TCCTCCGCTTATTGATATGC-3'.
The PCR amplification reaction system is as follows: DNA template (50 ng/. Mu.l) 0.5. Mu.l, 10mM 10 Xbuffer (Mg-containing Cl) 2 ) 2.5. Mu.l of Taq enzyme 0.2. Mu.l, 10. Mu.M ITS1 and ITS4 primers 0.5. Mu.l each, 10. Mu.M upstream and downstream primers 0.5. Mu.l each, dd H 2 The O-water was made up to a volume of 25. Mu.l.
The reaction procedure for PCR amplification was: pre-denaturation at 94℃for 4min; denaturation at 94℃for 45s, annealing at 55℃for 45s, elongation at 72℃for 1min,30 cycles; 72 min of extension and 4 ℃.
The 16S rDNA sequence of Trichoderma harzianum (Trichoderma harzianum) M6 is shown in SEQ ID NO. 1.
BLAST alignment results of the Trichoderma harzianum M6 sequence on NCBI show that Trichoderma harzianum M6 and other Trichoderma strains phylogenetic tree as shown in FIG. 2.
3.3 preservation of Trichoderma harzianum (Trichoderma harzianum) M6
Trichoderma harzianum (Trichoderma harzianum) M6 is preserved in China general microbiological culture Collection center (China center), with an address of Hospital No. 3, north Chen West Lu No.1, of the Korean region of Beijing, a preservation number of CGMCC No.40041, and a preservation date of 2022, 1 month and 11 days.
Example 2 biocontrol Effect of Trichoderma harzianum M6 on sunflower sclerotinia
1. Determination of nuclear capacity of trichoderma harzianum M6 parasitic sunflower sclerotinia sclerotiorum
1.1 Experimental methods
(1) Spore suspension preparation
Inoculating Trichoderma harzianum M6 strain on a PDA plate with glassine paper, culturing for 5 days, scraping spores with a small spoon, measuring spore concentration with a blood cell counting plate, and diluting with sterile water to 1×10 6 Is a spore suspension of (a).
(2) Sclerotium culture
The sclerotinia sclerotiorum SS-12 (separated and stored in the laboratory) is inoculated on a fresh potato-carrot (1:1) culture medium, and is placed in a constant temperature incubator at 25 ℃ for culture for 15 days, sclerotium is picked out and naturally dried for later use.
(3) Taking cultured sclerotium of sunflower with uniform size, sterilizing with 70% alcohol for 5min, washing with sterile water for 3 times, soaking sclerotium in suspension of Trichoderma for 30min, semi-immersing in culture dish containing sterilized fine sand, placing sclerotium 8-10 grains in each sand dish, taking sterile control, and repeating every treatment for 3 times. After incubation for 30d at 20 ℃, the parasitic peristalsis of the sclerotium was recorded.
(4) Sclerotium rot degree grading standard
Grade 0, no trichoderma reesei on the sclerotium surface; the surface of the sclerotium is provided with trichoderma hypha, but a large number of meristematic peduncles are not generated; 2, a large number of meristematic peduncles are arranged on the surface of the sclerotium, but the surface layer is not rotten; grade 3, the sclerotium surface layer is rotten and the internal color is changed to be soft; grade 4, complete sclerotium rot. And calculating the sclerotium rot index according to a disease index calculation formula.
Sclerotium disease rot index=e (number of sclerotium disease stage strains×number of disease stage representative value)/(total number of sclerotium) ×number of disease stage representative value) ×100
1.2 experimental results
The ability of Trichoderma harzianum M6 strain to parasitize sclerotium was determined by culturing sclerotium of spore liquid-soaked sclerotium of sclerotium SS-12 (FIG. 3) on a sterile sand table at 20℃for 20 d. The result shows that the Trichoderma harzianum M6 strain has strong parasitism capability (figure 4) on sclerotium of sunflower sclerotium, the parasitism rate reaches 98.3%, the sclerotium disease rot index is 88.3, and the inside of the sclerotium becomes soft and rot after 30 days (figure 5), which shows that the Trichoderma harzianum M6 strain has good biocontrol capability on sclerotium.
TABLE 1 indoor test of the effect of Trichoderma harzianum M6 on Nuclear parasitics of Helianthus
Figure BDA0003541399730000081
2. Antagonism of strains against Helianthus annuus
2.1 Experimental methods
(1) Counter experiment: from the colony edges of activated Trichoderma harzianum M6 strain and Sclerotinia sclerotiorum strain SS-12, mycelia were removed by a 5mm punch, and inoculated on PDA plates for counter culture. Wherein the distance between the trichoderma reesei mycelium pellet and the sclerotinia sclerotiorum mycelium pellet is 4cm, three repetitions are set for each treatment, and a flat plate only connected with the sclerotinia sclerotiorum mycelium pellet is used as a control for culture in an incubator at 20 ℃. The colony growth radii of trichoderma and sclerotinia are measured day by day, and whether the trichoderma colony covers the pathogenic bacteria colony or not is observed, and whether sclerotinia is generated or not is observed. When the control sclerotinia sclerotiorum grows to be full of 3/4 of a dish, the inhibition rate is calculated according to the following formula.
Inhibition = (control colony radius-treated colony radius)/control colony radius x 100
(2) Microscopic observation: 1ml of the melted sterilized PDA culture medium is sucked by a pipette, slowly dripped on a sterilized glass slide to prepare a PDA film with the thickness of about 1mm, and two ends of the glass slide are respectively inoculated with hypha blocks of trichoderma and sclerotinia sclerotiorum with the diameters of 5mm, and the trichoderma and the sclerotinia sclerotiorum are cultured for 1-3d at the temperature of 25 ℃ at the distance of 5cm, observed by an optical microscope and recorded.
2.2 experimental results
The result of the opposite culture of the Trichoderma harzianum M6 strain and the sclerotinia sclerotiorum SS-12 strain shows that when the two strains are not contacted, the growth of the sclerotinia sclerotiorum SS-12 strain is consistent with the growth of hyphae of a control strain, and the growth of the sclerotinia sclerotiorum SS-12 strain is not inhibited. After contact, growth of the sclerotinia sclerotiorum SS-12 strain was inhibited, eventually covered with trichoderma spores (fig. 6a, b); microscopic observation of the two strains after contact shows that Trichoderma harzianum M6 strains form hyphal branching to generate a ring structure to carry out ring winding on sclerotinia sclerotiorum SS-12 hyphae and parasitize the sclerotinia sclerotiorum hyphae (figure 6 c); after continuous culture, mycelium is rarefied and transparent at the contact position of the two strains, the yellow stripe has light color, and sclerotinia is not produced basically by the sclerotinia sclerotiorum SS-12 strain.
Example 3 preliminary application of Trichoderma harzianum M6 in controlling sclerotinia Helianthus
1. Sunflower seedling growth test
1.1 Experimental methods
Inoculating 1×10 strain by seedling method 6 The conidium suspension of the trichoderma harzianum M6 with individual spores/ml is inoculated to sunflower seedlings (sunflower variety is Solanum nigrum hybrid No. 8, the seedlings are planted in flowerpots with the diameter of 15ml, 10 plants are planted in each pot), 20ml of the conidium suspension is inoculated to each pot, the conidium suspension is inoculated for 3 times, 1 time every week is inoculated, 3 replicates are arranged in the treatment group, and meanwhile, a control group is arranged, and the sunflower seedlings of the control group are inoculated with equal amount of sterilized distilled water. After culturing in a greenhouse for 30 days, the whole plant is completely dug out, the plant height and root length are measured, the fresh weight (root fresh weight, stem leaf fresh weight) is measured, and the growth rate of the conidium suspension sunflower seedlings inoculated with Trichoderma harzianum M6 and the control group is calculated.
1.2 experimental results
After the sunflower seedlings are treated by the conidium suspension of trichoderma harzianum M6, the plant height, root length, root fresh weight, stem and leaf fresh weight and total fresh weight of the treated group are obviously higher than those of a control group (figure 7), and the root fresh weight, the stem fresh weight and the total fresh weight are respectively improved by 19.4%, 39.94% and 38.26% compared with the control group (table 2), so that the trichoderma harzianum M6 can promote the sunflower seedling growth.
TABLE 2 Trichoderma harzianum M6 promoting sunflower seedling growth effect
Figure BDA0003541399730000101
2. In vitro leaf anti-efficiency determination
2.1 Experimental methods
Picking sunflower leaf growing for 30 days, sterilizing with 70% alcohol,washing with sterile water; dilution of Trichoderma harzianum M6 conidium to 1X 10 6 Spraying spore suspension of individual spores/ml on the sunflower leaf surface; the control chemical agent uses 1000 times liquid of 45% dimethachlon water dispersible granule; for each treatment of 3 leaves, 3 replicates, the blank was sprayed with sterile water. After 24 hours, the center of the leaf is inoculated with a sclerotium rolfsii dish (the diameter is about 0.5 cm), the leaf is evenly placed in a plastic dish which is filled with double-layer gauze and is saturated by water and is sealed by a sealing film, then the plastic dish is placed in an illumination incubator for culturing for 16h/8h at the temperature of light/dark alternation, water is periodically added, the humidity in the dish is kept, and after obvious occurrence of blank control, the area of each leaf is measured.
Disease control effect (%) = (control spot diameter-treated spot diameter)/(control spot diameter) ×100% of control spot diameter
2.2 test results
As can be seen from table 3 and fig. 8, there was a significant difference between each treatment compared to the untreated control. When the average diameter of the disease spots of the control group is 5.5cm, the diameter of the clean disease spots of 45% of sclerotium of the chemical agent is 0.7cm, and the prevention effect is 86.7%; the diameter of the disease spot of the Harz raspberry M6 is 1.4cm, and the prevention effect is 73.9%, which indicates that the Harz raspberry M6 can effectively inhibit the infection of sclerotinia bacteria to sunflower leaves.
TABLE 3 determination of in vitro control efficacy of Harz raspberry M6 on Helianthus tuberosus
Figure BDA0003541399730000111
3. Determination of sclerotium parasitism in soil by Trichoderma harzianum M6
3.1 Experimental methods
Preparation of sunflower sclerotium: after activation culture, the sclerotinia sclerotiorum strain is inoculated on a sterilized potato-carrot (1:1) culture medium, and is subjected to dark culture in an incubator at 18-20 ℃ for 4 weeks, after black hard sclerotium is produced, the sclerotium is washed out by clear water, and the sclerotium is dried in the shade and stored for standby.
The cultured sclerotium was put in a gauze and buried in a flowerpot (sclerotium depth: 5 cm) containing soil, and then 20ml of the gauze containing 1X 10 6 The conidium suspension of Trichoderma harzianum M6, which is a single spore/ml, was poured into soil evenly, and a control group was inoculated with sterile water, and the rotting index of the sclerotium was investigated after 30 days for each treatment of 30 sclerotium, 3 replicates (investigation method was referred to 1.1).
3.2 experimental results
The parasitic rot ability of the Trichoderma harzianum M6 strain on the sclerotium of the sunflower in the soil is measured through a potting test, and the result shows that after 30 days, the disease rot index of the control sclerotium is 0, the parasitic rate of the Trichoderma harzianum M6 strain of the treatment group on the sclerotium in the soil is 95.6%, the disease rot index of the sclerotium is 74.7, and most sclerotium is decomposed (figure 9), so that the Trichoderma harzianum M6 strain has good parasitic rot ability on the sclerotium of the sunflower.
TABLE 4 parasitism of Trichoderma harzianum M6 on sclerotium of sunflower sclerotium (potting test)
Figure BDA0003541399730000121
4. Trichoderma harzianum M6 control sunflower seedling sclerotinia sclerotiorum potting test
4.1 Experimental methods
Uniformly mixing sunflower sclerotium and soil, putting into a flowerpot with the diameter of 15ml, ensuring that 50 sclerotium is contained in each pot, sowing a proper amount of sunflower seeds in each flower disc, keeping 10 seedlings on each disc, placing in a greenhouse for normal growth, watering the sunflower every 2 days, and performing subsequent experiments until two pairs of true leaves can grow out of the cultured sunflower.
The conidium suspension of the experimental Trichoderma harzianum M6 (1X 10) 6 Individual spores/ml) 20ml were evenly poured into the flower disc, 7 days later, 1 time of trichoderma harzianum M6 spore suspension was poured, 50% carbendazim and sterile water as chemical agents were used as a control group, 3 times of repetition were performed per treatment of the 3 discs, and when the control group was fully developed, the incidence of sunflower sclerotinia was investigated, and the control effect was calculated.
4.2 experimental results
As can be seen from table 5 and fig. 10, there was a significant difference between each treatment compared to the untreated control. When the average morbidity of the control group is 74.4%, the average morbidity of the chemical agent 50% carbendazim is 16.7%, the prevention effect is 77.6%, the average morbidity of the Harz raspberry M6 is 20.7%, the prevention effect is 72.1%, and the morbidity of the two is not significantly different, so that the Harz raspberry M6 has significant prevention and treatment effects on sunflower seedling rot.
TABLE 5 prevention and treatment effect of Trichoderma harzianum M6 on sunflower seedling rot type sclerotium disease (potted plant test)
Figure BDA0003541399730000131
While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Figure BDA0003541399730000141
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Sequence listing
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<120> Trichoderma harzianum M6 for preventing and treating sclerotinia rot of sunflower and application thereof
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Claims (6)

1. Trichoderma harzianumTrichoderma harzianum) M6, the preservation number of which in China general microbiological culture Collection center is CGMCC No.40041.
2. A microbial inoculum comprising the Trichoderma harzianum strain according to claim 1 as an active ingredientTrichoderma harzianum)M6。
3. A biocontrol agent for promoting sunflower growth, which comprises the Trichoderma harzianum strain of claim 1 as an active ingredientTrichoderma harzianum) M6 or the microbial inoculum of claim 2.
4. The biocontrol agent of claim 3, wherein,
the promotion of sunflower growth is manifested by an increase in sunflower plant height or an increase in sunflower biomass.
5. The Trichoderma harzianum strain of claim 1Trichoderma harzianum) Use of M6 or the biocontrol formulation of claim 3 to promote sunflower growth.
6. The use according to claim 5, characterized in that,
the promotion of sunflower growth is manifested by an increase in sunflower plant height or an increase in sunflower biomass.
CN202210240527.6A 2022-03-04 2022-03-10 Trichoderma harzianum M6 for preventing and treating sclerotinia sclerotiorum of sunflower and application thereof Active CN114480143B (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2485796A1 (en) * 2004-06-18 2005-12-18 Thomas D. Johnson Controlling plant pathogens with fungal/bacterial antagonist combinations comprising trichoderma virens and bacillus subtilis var.amyloliquefaciens
CN104830700A (en) * 2015-05-06 2015-08-12 黑龙江省农业科学院植物保护研究所 Trichoderma sp efficient spore-production medium and culture method thereof
CN106754536A (en) * 2016-12-31 2017-05-31 中国农业科学院农业资源与农业区划研究所 Bacillus circulans Y3 and its application in soil-borne disease of crop is prevented and treated

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2485796A1 (en) * 2004-06-18 2005-12-18 Thomas D. Johnson Controlling plant pathogens with fungal/bacterial antagonist combinations comprising trichoderma virens and bacillus subtilis var.amyloliquefaciens
CN104830700A (en) * 2015-05-06 2015-08-12 黑龙江省农业科学院植物保护研究所 Trichoderma sp efficient spore-production medium and culture method thereof
CN106754536A (en) * 2016-12-31 2017-05-31 中国农业科学院农业资源与农业区划研究所 Bacillus circulans Y3 and its application in soil-borne disease of crop is prevented and treated

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