CN115975821A - Trichoderma africanum Tr35 and application thereof - Google Patents
Trichoderma africanum Tr35 and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biological control and discloses Trichoderma africanum Tr35 and application thereof. The Trichoderma africanum Tr35 is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC NO.40207. The Trichoderma africanum Tr35 has the function of broad-spectrum antagonism of plant pathogenic fungi, and can be used for disease control of grain crops, economic crops, medicinal plants and the like; the strain has a good effect of promoting the growth and development of plants, and has a promoting effect on the germination and root growth of corn seeds.
Description
Technical Field
The invention belongs to the field of biological control, and particularly relates to Trichoderma africanum Tr35 and application thereof.
Background
The corn stalk rot is an important disease commonly occurring in world corn production areas and is seriously occurring in various corn planting areas in China. In general years, the field plant disease rate is 5% -10%, and when serious diseases occur, the field plant disease rate can reach more than 50%. The annual yield loss in reoccurrence is 5-10%, and the yield of serious plots is reduced by about 25%. The corn stalk rot pathogen has complex composition, and more than 20 kinds of corn stalk rot pathogens are reported, and the corn stalk rot pathogens are singly or compositely infected to cause the corn stalk rot, wherein the main pathogens of the corn stalk rot disease in China are Fusarium sp. There are more than 15 species of fusarium that are identified as pathogenic bacteria, among which fusarium graminearum (f.graminearum), fusarium verticillioides (f.verticillioides), and fusarium laminarinum (f.proliferatum) are the 3 pathogenic fusarium species with the highest frequency of isolation. In the prior production, the prevention and the treatment of the disease mainly adopt a chemical seed coating agent containing fludioxonil and an additional potassium fertilizer, but the prevention effect is mostly only about 30 percent. Meanwhile, the problems of pesticide residue, environmental pollution, drug resistance of diseases and pests, destruction of farmland micro-ecological balance, threat to biological health and the like caused by long-term and large-scale use of chemical pesticides are increasingly highlighted.
Biological control is to take microorganism with disease prevention and growth promotion functions as a biological control factor to control plant diseases. In recent years, the safety and high efficiency of the plant are gradually emphasized, and the plant is an important measure for replacing chemical prevention and control. Trichoderma (Trichoderma) is a biocontrol fungus widely distributed in nature, has antagonism on at least more than 18 pathogenic fungi and more than 20 pathogenic bacteria, is mainly used for preventing and treating soil-borne diseases of various plants and partial leaf and spike diseases, and has higher economic value and application prospect.
Trichoderma atroviride SG3403 (patent CN 103484377A), trichoderma asperellum ZJSX5003 (patent CN 103484376A) and Trichoderma harzianum LTR-2 (patent CN 11081074A) which are currently used for preventing and controlling corn stalk rot, but the Trichoderma atroviride strains only effectively prevent and control the stalk rot caused by Fusarium graminearum, and have narrow prevention and control spectrum and unobvious prevention and control effect. In patent CN11081074A, although the control effect of trichoderma harzianum on corn stalk rot is described as 72.53%, the incidence of disease is very low, only 1.02% and 3.72% based on the control group and the treatment group described in the specification, and the statistical control effect data can be calculated as invalid results, which is not of reference value. Therefore, it is particularly important to provide a novel strain having a wider control spectrum and having a control effect on various plant diseases.
Disclosure of Invention
In view of this, the invention provides a Trichoderma africanum (Trichoderma africanum) Tr35 with broad-spectrum antagonistic plant pathogenic bacteria effect, and the Trichoderma africanum Tr35 of the invention has the effects of preventing and treating various plant diseases and promoting plant growth and development.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides Trichoderma africanum which is Trichoderma africanum Tr35 with the preservation number of CGMCC NO.40207.
In the invention, the EF-1 alpha gene sequence of the Trichoderma africanum Tr35 is shown as SEQ ID NO. 1, and the RPB2 gene sequence is shown as SEQ ID NO. 2.
In the invention, the morphological characteristics of the trichoderma africanum Tr35 are as follows: the colony is round, white at the initial stage and gradually turns into green; hypha is slender and flocculent and grows radially from the center to the periphery; conidiophores are in a shape from a flask to an ampoule, the middle part is expanded, the top end is finest and can produce spores, and cells of the conidiophores are single or generate 2-5 rounds; the conidium is spherical or ellipsoidal, has single cell, smooth surface, light green color, and deep green color after aging.
The present invention also includes a culture of the above Trichoderma africanum, which is obtained by culturing the above Trichoderma africanum Tr35 in a microbial culture medium.
As an alternative embodiment, the culture comprises a bacterial suspension, spore suspension, fermentation broth of Trichoderma africanum Tr35 and/or metabolites thereof.
It is a further object of the present invention to provide the use of Trichoderma africanum Tr35 and/or a culture thereof as described above in any one of:
1) Inhibiting phytopathogens; the plant pathogenic bacteria are at least one of the following: fusarium graminearum, fusarium oxysporum, fusarium verticillium, fusarium sporogenes, fusarium cerealis, fusarium crescent moon, helminthosporium cerealis, rhizoctonia cerealis, fusarium graminearum, fusarium solani and Alternaria ginseng;
2) Preparing 1) an inhibitor of said phytopathogen;
3) Controlling 1) plant diseases caused by said phytopathogens;
4) Preparing 3) the inhibitor of plant diseases.
Preferably, the plant disease includes at least one of: corn stalk rot, corn ear rot, corn root rot, corn sheath rot, corn small leaf spot, curvularia leaf spot, wheat root rot leaf spot, wheat sheath blight, wheat root rot, wheat basal rot, wheat scab, soybean root rot, ginseng black spot, and tobacco root rot.
As an alternative embodiment, the Trichoderma africanum Tr35 is used at a concentration of 10 6 ～10 8 spores/mL.
It is a further object of the present invention to provide the use of Trichoderma africanum Tr35 and/or a culture thereof as described above in any one of:
a) Promoting the growth of plants;
b) Preparing a product for promoting plant growth;
c) Promoting the germination of plant seeds;
d) Preparing the product for promoting the germination of the plant seeds.
As one possible embodiment, the plant comprises corn and the promoting plant growth comprises promoting the growth of the embryo, radicle, and lateral roots of corn.
The invention also provides a microbial agent or fertilizer, which contains the Trichoderma africanum Tr35 and/or culture.
Compared with the prior art, the invention has the following advantages and effects:
the Trichoderma africanum Tr35 has the broad-spectrum antagonistic action on plant pathogenic fungi, and can be used for disease control of grain crops, economic crops, medicinal plants and the like; the strain has a good effect of promoting the growth and development of plants, and has a promoting effect on the germination and root growth of corn seeds.
Deposit description
Trichoderma africanum Tr35 was deposited in the China general microbiological culture Collection center (CGMCC) at 2022, 6.2, with the deposit number of CGMCC NO.40207; address: xilu No. 1, beijing, chaoyang, beijing, and institute for microbiology, china academy of sciences.
Drawings
FIG. 1 shows a Tr35 strain phylogenetic tree obtained from the EF-1. Alpha. Gene sequence.
FIG. 2 is a Tr35 strain phylogenetic tree obtained from the sequence of the RPB2 gene.
Detailed Description
The Trichoderma africanum (Trichoderma afraharizianum) Tr35 is obtained by separating and purifying rhizosphere soil of a corn stalk rot disease strain in Zhou city, henan province, and the strain has broad-spectrum plant pathogenic fungus antagonism and plant growth promotion effects.
At present, the African Trichoderma harzianum has been preserved in China general microbiological culture Collection center, with the preservation number: CGMCC NO.40207; address: beijing, haoyang district, xilu No. 1, ministry of microbiology, china academy of sciences, 3; the preservation date is as follows: 2/6/2022, and is classified and named as Trichoderma afraharizia Tr35.
The morphological characteristics of the Trichoderma africanum Tr35 are as follows: the colony is round, white at the initial stage and gradually turns into green; hypha is slender and flocculent and grows radially from the center to the periphery; conidiophores are in a shape from a flask to an ampoule, the middle part is expanded, the top end is finest and can produce spores, and cells of the conidiophores are single or generate 2-5 rounds; the conidium is spherical or ellipsoidal, has single cell, smooth surface, light green color, and deep green color after aging.
The EF-1 alpha gene sequence of the Trichoderma africanum Tr35 is shown as SEQ ID NO. 1, and the RPB2 gene sequence is shown as SEQ ID NO. 2.
Trichoderma africanum Tr35 was identified as a Trichoderma fungus (T. Afroharizianum) of Deuteromycotina, class Hyphomycetes, order Hyphomycetales, genus myxosporium.
In the present invention, the culture of Trichoderma africanum refers to a culture obtained by culturing Trichoderma africanum Tr35 in a microbial culture medium, and includes, but is not limited to, a bacterial suspension, a spore suspension, a fermentation broth and/or a metabolite thereof of Trichoderma africanum Tr35. The microorganism culture medium refers to a culture medium capable of realizing the growth and propagation of the Trichoderma africanum Tr35, and includes but is not limited to a PDA culture medium, and a modified culture medium on the PDA culture medium for realizing the better growth or metabolism of the Trichoderma africanum Tr35. The medium and the culture conditions can be adjusted or modified by those skilled in the art according to the actual culture conditions.
The Trichoderma africanum Tr35 and the culture thereof have the function of inhibiting various phytopathogens and can be used as the only or partial active component of the phytopathogen inhibitor. Preferably, the trichoderma africanum Tr35 and the culture thereof of the present invention have inhibitory effects on various plant pathogenic fungi such as Fusarium graminearum (Fusarium graminearum), fusarium oxysporum (f.oxysporum), fusarium verticillioides (f.verticillioides), fusarium laminarinum (f.proliferum), fusarium solani (Bipolaris maydis), fusarium lunatum (Curvularia lunata), fusarium verticillium dahliae (Bipolaris sorokiniana), rhizoctonia cerealis (Rhizoctonia cerealis), fusarium pseudograminearum (f.pseudograminearum), fusarium solani (f.sorangium), alternaria ginseng (Alternaria panax).
The Trichoderma africanum Tr35 and the culture thereof can prevent and control plants by inhibiting phytopathogensThe plant disease inhibitor can be used as the only or partial active ingredient of the plant disease inhibitor. As one embodiment, the plant diseases of the present invention include at least one of: corn stalk rot, corn ear rot, corn root rot, corn sheath rot, corn small leaf spot, curvularia leaf spot, wheat root rot leaf spot, wheat sheath blight, wheat root rot, wheat stalk rot, wheat scab, soybean root rot, ginseng black spot, and tobacco root rot. As an alternative embodiment, the Trichoderma africanum Tr35 is used at a concentration of 10 6 ～10 8 spores/mL. Experiments prove that the Trichoderma africanum Tr35 gene of the invention is 10 6 Under the using concentration of each spore/mL, the composition has good control effects on fusarium graminearum stem rot, fusarium verticillioides stem rot and fusarium laminarum stem rot, the control effect is more than 76.88 percent, and the in vitro control effect on corn small leaf spot is 87.92 percent.
The Trichoderma africanum Tr35 and the culture thereof have the effect of promoting plant growth and can be used as the only or partial active component of a product for promoting plant growth. The Trichoderma africanum Tr35 and the culture thereof have the effect of promoting the germination of plant seeds, and can be used as a unique or partial active ingredient of a product for promoting the germination of the plant seeds. In one embodiment, trichoderma africanum Tr35 and cultures thereof of the present invention are capable of promoting germination of maize, and growth of embryos, radicles and lateral roots. In one embodiment, the seed is soaked in the fermentation broth of trichoderma africanum Tr35 in the form of a stock solution or a dilution, optionally with a dilution factor of 1 to 300, more preferably 2 to 200, or any dilution factor in the above range, such as 2, 10, 30, 50, 100, 150, 200.
In another aspect of the present invention, there is provided a microbial agent or fertilizer comprising Trichoderma africanum Tr35 and/or a culture thereof according to the present invention. The microbial agents or fertilizers of the invention can be produced according to methods well known to those skilled in the art. The preparation type of the microbial agent or the fertilizer can be in the forms of solution, dispersing agent, suspending agent, granules and the like. The application method can be spraying, broadcasting, furrow application or irrigation with water.
In one embodiment, the microbial agent or the fertilizer further comprises a microorganism type different from that of Trichoderma africanum Tr35, and different microorganisms are compounded for use, so that the host spectrum of the microbial agent is expanded, the control effect on plant pathogenic bacteria is improved, and the generation of drug resistance is delayed. The microbial fertilizer of the invention can also comprise organic and inorganic fertilizers required by plant growth. The African trichoderma harzianum Tr35 or the culture thereof and the organic and inorganic fertilizer are jointly made into the microbial fertilizer, so that the growth promoting effect of the microbial fertilizer is improved while the plant diseases are prevented and treated.
In one embodiment, the trichoderma africanum Tr35 or culture thereof of the present invention may be freeze-dried or dissolved in a suitable vehicle to prepare microbial agents or fertilizers. Lyophilization is carried out using methods conventional in the art. The solvent can be selected from water, a microorganism culture medium and the like.
The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples. The phytopathogens used in the examples below were derived from laboratory-stored phytopathogen species and were of known type. The experimental methods used in the examples are conventional methods unless otherwise specified, and the materials, reagents and the like used therein are commercially available.
Example 1
Isolation and purification of Trichoderma strains
In the mature period of the corn, a five-point sampling method is adopted, soil samples of 10-20 cm of plough layer are collected from the periphery of corn stalk rot disease plants in a field with corn stalk rot disease, the soil samples in the same community are uniformly mixed, numbered and filled into a freshness protection package, and the freshness protection package is taken back to a laboratory for storage at 4 ℃. And collecting 124 parts of soil sample.
By dilution plate method, 10g of soil sample is taken from refrigerator and put into triangular flask containing 90ml of sterilized water and 5-10 glass beads with diameter of 4mm, and then shaken on shaking table of 200r/min for 30min. Pouring 5ml into a triangular flask containing 45ml of sterilized water, mixing uniformly, sucking 5ml and transferring into the triangular flask containing 45ml of sterilized water, sucking 0.2ml after mixing uniformly on a flat plate with the diameter of 9cm, coating uniformly by using a coater, and culturing in a constant-temperature incubator at 28 ℃. And after 48-72 h, selecting a bacterial colony producing green conidia, transferring the bacterial colony to a PDA (personal digital assistant) plate, continuously culturing in a constant-temperature incubator at 28 ℃, and transferring the bacterial colony to the PDA plate again for constant-temperature culture at 28 ℃ when the diameter of the bacterial colony is 4-5 cm. A total of 109 Trichoderma strains were obtained.
The plate confronting method is adopted to test the inhibition effect of the trichoderma strains on the fusarium stalk rot pathogenic bacteria of corn. Activating main pathogenic bacteria of fusarium graminearum, fusarium verticillioides and fusarium laminae stored in a laboratory, respectively culturing the activated pathogenic bacteria and trichoderma on a PDA (personal digital assistant) plate for 5 days, respectively beating fungus cakes with the diameter of 0.5cm on the edges of bacterial colonies by using a puncher to face each other and inoculating the fungus cakes on two sides of the PDA plate with the diameter of 9cm, wherein the distance between the centers of the fungus cakes is about 4.5cm, and connecting lines pass through the center of the plate and are cultured in a constant-temperature incubator at 28 ℃. The inoculated Fusarium graminearum, fusarium verticillium and Fusarium stratiotes were also grown in pure culture as controls, and repeated 3 times per treatment. The colony diameter at 5d was measured and recorded by the cross method, and the inhibition rate was calculated.
Inhibition rate = (diameter of colony of control pathogen-diameter of colony of treated pathogen)/(diameter of colony of control pathogen-0.5 cm) × 100%.
As can be seen from Table 1, trichoderma strain Tr35 has high inhibitory effects on Fusarium graminearum, fusarium verticillium and Fusarium cambium, and the inhibitory rates are 76.41%, 72.42% and 73.74%, respectively.
TABLE 1Tr35 determination of inhibition rate of main pathogenic bacteria of fusarium stem rot of maize
Example 2
Morphological characterization of Tr35 Strain
A cake of 0.5cm in diameter was punched out of the edge of the colony of the Tr35 strain by a punch and placed on a PDA plate and cultured in the dark at 28 ℃. The growth rate, color change, and other characteristics of the colonies on the petri dish were observed daily. After the bacterial colony is cultured for 3-5 days, selecting hypha and making into slices, and observing the shapes of the hypha, conidia and spore stalks under an optical microscope.
As a result: after the Tr35 strain is activated on the PDA culture medium, aerial hyphae grow rapidly. The colony was round, white in the early stage and gradually turned green. The hyphae are filamentous, slender and flocculent, and grow radially from the center to the periphery. Conidiophores are in a shape from a flask to an ampoule, the middle of the conidiophores is expanded, the top of the conidiophores is finest, spores can be produced, and cells of the conidiophores grow singly or grow in 2-5 rounds. The conidium is spherical or ellipsoidal, has single cell, smooth surface, light green color, and dark green color after aging. Through the morphological characteristics of the strains, the strains are preliminarily identified to be one of Trichoderma (Trichoderma).
Example 3
Molecular characterization of Tr35 strains
Culturing Trichoderma strain Tr35 in PDA culture medium for 5 days, collecting spore and mycelium, and extracting total DNA of fungi by CTAB method.
Respectively carrying out PCR amplification by using EF-1 alpha gene primers and RPB2 gene primers. The primers were synthesized by Shanghai Bioengineering Co., ltd.
EF-1 alpha gene primers are as follows:
EF1-728F：5'-CATCGAGAAGTTCGAGAAGG-3'；
TEF1LLErev：5'-AACTTGCAGGCAATGTGG-3'。
the RPB2 gene primers were as follows:
5F：GAYGAYMGWGATCAYTTYGG；
7CR：CCCATRGCTTGYTTRCCCAT。
the PCR reaction system was 25. Mu.L: wherein 12.5. Mu.L 2. Mu.Es Taq MasterMix (kang century), 1. Mu.L template DNA, 0.5. Mu.L each of forward and reverse primers (10. Mu. Mol/L), ddH 2 Make up to 25. Mu.L of O.
PCR amplification reaction procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s, annealing at 53 ℃ (primers EF1-728F and TEF1 LLErev) and 51 ℃ (primers 5F and 7 CR) respectively, annealing for 30s, extension at 72 ℃ for 1min, and 35 cycles; finally, extension was carried out at 72 ℃ for 7min.
The PCR amplification product is directly delivered to Shanghai biological engineering GmbH for sequencing. And (3) carrying out homology comparison in GenBank according to a sequencing result, and constructing a phylogenetic tree by utilizing MEGA5.2 software according to the obtained sequence and other pathogenic bacteria related sequence information obtained from the GenBank to determine the classification status of the pathogenic bacteria.
The EF-1 alpha and the RPB2 of the strain Tr35 are subjected to PCR amplification, and the sizes of amplification products are 1276bp and 1136bp respectively. Sequencing the amplified product, wherein the sequencing result is shown as SEQ ID NO. 1 and SEQ ID NO. 2. The obtained EF-1 alpha gene sequence and the RPB2 gene sequence of the Tr35 strain are subjected to homology comparison in GenBank, and the result shows that the strain Tr35 has the closest affinity relationship with the EF-1 alpha gene sequence of the African Trichoderma harzianum strain Z19, and the homology is 99.84 percent; the homology is 99.74% and 99.91% respectively, which is closest to the relative relationship of the RPB2 gene sequences of Trichoderma Africa strain Tri-1 and Tafum 2. The MEGA5.2 software was used to construct a phylogenetic tree, and the results showed that the strain Tr35 was aggregated with Trichoderma africanum, indicating that Tr35 belongs to Trichoderma africanum (T. Afroharizanum).
According to the culture character, morphological characteristics, EF-1 alpha gene sequence and RPB2 gene sequence analysis result of the strain Tr35, the strain is identified as Trichoderma fungus of Deuteromycotina, hyphomycetes and myxomycete, and T.african Harzianum.
Example 4
Determination of the bacteriostatic Spectrum of Tr35
Adopting a plate confronting method, respectively carrying out confronting culture on the strain Tr35 on a PDA culture medium and strains of corn ear rot, corn leaf spot, curvularia leaf spot, corn root rot, corn sheath rot, wheat root rot, wheat sharp leaf blight, wheat stem rot, wheat gibberellic disease, soybean root rot, ginseng black spot and tobacco root rot which are main pathogens of corn, carrying out 28 ℃ dark culture, measuring the growth diameter of a bacterial colony by a cross method after the comparison group of bacterial filaments are full of the culture medium, repeating the steps for 3 times by taking the PDA culture medium only inoculated with the pathogenic bacteria as a comparison, and calculating the inhibition rate of the strain Tr35 on the growth of the pathogenic bacteria.
The test results show that (table 2), the Trichoderma africanum strain Tr35 has good inhibition effect on the growth of 14 tested plant pathogenic fungi hyphae, the inhibition rate reaches over 60 percent, and the Trichoderma africanum strain Tr35 has a wide antifungal spectrum. Wherein the inhibition rate on Rhizoctonia cerealis reaches 84.41%, and the inhibition rates on hypha of corn microsporum, ginseng black spot, wheat root rot and ear rot are all more than 70%, and are respectively 75.56%, 74.63%, 74.14%, 74.00% and 71.11%.
TABLE 2Tr35 inhibition of growth of pathogenic fungi colonies on test plants
Example 5
Effect of Tr35 on maize growth and development
Tr35 was inoculated on a PDA plate, cultured at 28 ℃ for 4-7 days in the dark, and when colonies grew sufficiently, 10 cakes (diameter 0.5 cm) were excised from the edge, inoculated in a PD liquid medium (100 mL PD/250mL Erlenmeyer flask), and cultured at 28 ℃ for 5 days with shaking at 180 r/min. Filtering to remove mycelium to obtain fermentation liquor. Preparing the fermentation liquor into 1 × 10 7 Spore liquid per ml, diluted by the following fold respectively: 2 times, 10 times, 50 times, 100 times and 200 times. Soaking semen Maydis in the above diluent for 3 hr, air drying, placing into sterilized culture dish with filter paper, culturing at 28 deg.C, adding sterile water after 48 hr, culturing at 28 deg.C, repeating the above steps for 3 times, and repeating 10 semen Maydis each time. After 5d, the germination rates of the different treatments were investigated and the germ, radicle length and lateral root number were measured.
The test results show that (table 3): after the Tr35 fermentation liquor is treated by 10 times of diluent, the germination rate of the corn is the highest and is 100%, and the promotion effect on the growth of corn germs, radicles and lateral roots is most obvious, and the obvious difference level is achieved compared with the contrast.
TABLE 3 influence of Tr35 fermentation broths of different concentrations on corn growth
Example 6
Tr35 control effect on fusarium zeae stalk rot
Tr35 fermentation broth was prepared by the method of example 5. Filtering to remove mycelium, and diluting spore concentration to 1 × 10 6 one/mL for use. Selecting healthy Zhengdan 958 seeds with consistent size, sterilizing with 75% alcohol for 30s, sterilizing with 2% sodium hypochlorite for 10min, and washing with sterile water for 3 times for use. Soaking sterilized seed in the obtained Tr35 spore fermentation diluent for 3h, air drying, sowing in sterile soil, and taking Zhengdan 958 seed soaked in sterile water as control. Inoculating Fusarium graminearum, fusarium verticillium and Fusarium stratiotes to the grown 2-leaf 1-heart strain at a concentration of 2 × 10 6 Per ml, 5ml of root irrigation is carried out on each plant. After 10 days, observing the infection condition of pathogenic bacteria, investigating the morbidity and calculating the control effect.
Grading standard of stem rot in seedling stage: level 0: the whole plant grows normally and has no disease grade; stage 1: the underground part of the overground part of the corn grows basically normally, the area of disease spots accounts for the total area of the root surface, and the root group is white and brown; and 2, stage: obviously hindering overground and underground growth, lightening leaf color, only having plant height of 3/4 of that of the contrast, having few and short lateral roots, no fibrous roots and scab connected pieces, wherein the scab area accounts for 1/4-1/2 of the total area of the root surface, and the root group has the same color of white and brown; and 3, stage: the overground and underground parts grow extremely abnormally, the overground and underground parts can be seen to be withered and yellow-withered, the plant height is only 1/2 of that of the contrast, the lateral roots are extremely small, scabs account for 1/2-3/4 of the total area of the root surface, and the root groups are brown and white; 4, level: sprouting but not sprouting, almost suffocating, disease spots accounting for more than 3/4 of the total area of the root surface, and brown root.
The calculation method of the disease index and the prevention effect is as follows:
disease index =100 × ∑ (number of diseased plants at each stage × representative value at each stage)/(number of investigated total plants × highest representative value);
control effect (%) = (blank control disease index-treatment disease index)/blank control disease index.
The test results show (table 4): tr35 has better control effect on the stalk rot caused by 3 kinds of fusarium, the control effect reaches more than 70%, wherein the control effect on the stalk rot of fusarium graminearum caused by cereal fusarium reaches 81.99%, and the control effects on the stalk rot of fusarium verticillioides caused by pseudoverticillium and the stalk rot of fusarium exserotinatum are equivalent to 77.44% and 76.88%, respectively.
TABLE 4Tr35 Effect on Fusarium zeae Stem rot control
Example 7
Tr35 in-vitro control effect test on corn small leaf spot
Tr35 broth was prepared using the method of example 5. Filtering to remove mycelium, and diluting spore concentration to 1 × 10 6 one/mL for use. The in vitro leaf method is used for determining the prevention and treatment effect: selecting healthy corn leaves with consistent growth, and washing the leaves clean by using sterile water. At a spore concentration of 1X 10 6 Soaking in fermentation liquor per mL for 30min, wrapping two ends of each leaf with absorbent cotton, placing in culture dishes padded with sterile wet filter paper, placing one leaf in each culture dish, sealing with preservative film, and culturing at 28 deg.C for 1d. Inoculating cultured plaque bacteria cake on both sides of main leaf vein of leaf, sealing again, and culturing at 28 deg.C. Observing every day, measuring the diameter of the lesion spots by adopting cross phase multiplication after the contrast is fully developed, and calculating the area of the lesion spots. Blank controls were run and 6 replicates of each treatment were run.
Area of lesion (mm) 2 ) = lesion length (mm) × lesion width (mm) × 3.14/4.
Control effect (%) = (control lesion area-treatment lesion area)/control lesion area × 100.
Test resultsShows (Table 5): the lesion area of the leaf treated by the Tr35 fermentation liquor is 9.68mm 2 Significantly lower lesion area than the blank control (80.14 mm) 2 ) The control effect of the Tr35 fermentation broth on the corn northern leaf blight is 87.92%. The invention shows that the Trichoderma africanum strain Tr35 and the fermentation liquid thereof have outstanding control effect on the small spot.
TABLE 5Tr35 Ex vivo test for controlling northern corn leaf blight
The above embodiments are the best mode for carrying out the invention, but the 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 invention should be construed as equivalents thereof, and they are intended to be included in the scope of the invention.
Claims (10)
1. Trichoderma africanum is Trichoderma africanum Tr35 with the collection number CGMCC NO.40207.
2. The Trichoderma africanum of claim 1 wherein the EF-1 α gene sequence of Trichoderma africanum Tr35 is as shown in SEQ ID NO 1 and the RPB2 gene sequence is as shown in SEQ ID NO 2.
3. The culture of Trichoderma africanum according to claim 1 or 2, wherein the culture is a culture obtained by culturing Trichoderma africanum Tr35 according to any one of claims 1 to 3 in a microbial culture medium.
4. The culture according to claim 3, wherein the culture comprises a bacterial suspension, spore suspension, fermentation broth of Trichoderma africanum Tr35 and/or metabolites thereof.
5. Use of trichoderma africanum Tr35 according to claim 1 or 2 and/or of the culture according to claim 3 or 4 in any one of:
1) Inhibiting phytopathogens; the plant pathogenic bacteria are at least one of the following: fusarium graminearum, fusarium oxysporum, fusarium verticillium, fusarium sporogenes, fusarium cerealis, fusarium crescent moon, helminthosporium cerealis, rhizoctonia cerealis, fusarium graminearum, fusarium solani and Alternaria ginseng;
2) Preparing 1) an inhibitor of said phytopathogen;
3) Controlling 1) plant diseases caused by said phytopathogens;
4) Preparing 3) the inhibitor of plant diseases.
6. Use according to claim 5, wherein the plant diseases comprise at least one of: corn stalk rot, corn ear rot, corn root rot, corn sheath rot, corn small leaf spot, curvularia leaf spot, wheat root rot leaf spot, wheat sheath blight, wheat root rot, wheat basal rot, wheat scab, soybean root rot, ginseng black spot, and tobacco root rot.
7. The use according to claim 5, wherein Trichoderma africanum Tr35 is used at a concentration of 10 6 ～10 8 spores/mL.
8. Use of trichoderma africanum Tr35 according to claim 1 or 2 and/or a culture according to claim 4 or 5 in any one of:
a) Promoting the growth of plants;
b) Preparing a product for promoting plant growth;
c) Promoting the germination of plant seeds;
d) Preparing the product for promoting the germination of the plant seeds.
9. The use of claim 8, wherein the plant comprises corn and the promoting plant growth comprises promoting the growth of the embryo, radicle, and lateral roots of corn.
10. A microbial preparation or fertilizer comprising Trichoderma africanum Tr35 of any one of claims 1 to 3, and/or a culture of claim 4 or 5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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