CN112143654B - Trichoderma pseudokoningii and application thereof in preventing and treating litchi anthracnose - Google Patents
Trichoderma pseudokoningii and application thereof in preventing and treating litchi anthracnose Download PDFInfo
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Abstract
The invention belongs to the technical field of biological control of plant diseases, and discloses trichoderma pseudokoningii and application thereof in control of litchi anthracnose. The strain is named as Trichoderma pseudokoningii TJ5, and is preserved in Guangdong province microbial culture collection center, GDMCC for short, 8.8.2020, address: the microbial research institute of Guangdong province, No. 59 building, No. 5 building, of Mieli Zhonglu, Guangzhou City, the number of which is GDMCC No. 61129. The strain is antagonistic bacteria screened from litchi, and because the antagonistic bacteria coexists with litchi for a long time and symbioses with the litchi, a series of survival mechanisms similar to the antagonistic bacteria are formed, and the strain is used for preventing and treating litchi diseases, so that the strain is safer and more reliable for eating litchi and processing products of the litchi; the strain has an excellent inhibitory effect on litchi anthracnose pathogen, has strong bacteriostatic ability, and has important practical significance for preventing litchi anthracnose.
Description
Technical Field
The invention belongs to the technical field of biological control of plant diseases, and particularly relates to trichoderma pseudokoningii and application thereof in control of litchi anthracnose.
Background
Litchi chinensis Sonn is a subtropical evergreen plant and also an important subtropical commercial crop, belonging to Sapindaceae. Litchi in China is originally produced in south regions including provinces such as Guangdong, Guangxi, Fujian and Hainan. Litchi is a good product in fruits, has juicy pulp, is aromatic in flavor, tender and tasty, has the beauty of Chinese precious fruits for a long time, and has extremely high commercial value. However, the ripening of litchi fruits is mostly in high-temperature and high-humidity seasons, so that the picked fruits are easy to brown, rot and deteriorate, and the litchi anthracnose is a main cause of the rot and deterioration of the litchi fruits. Litchi anthracnose is a very important fungal disease in litchi production, and can cause huge economic loss before and after harvesting. At present, chemical control is mainly used for controlling litchi anthracnose, and although the chemical control can achieve certain effect in a short time, the continuous use of chemical agents is easy to increase the drug resistance of pathogenic bacteria and even the drug resistance, and simultaneously, the problems of pesticide residue, environmental pollution and the like are also accompanied. The biological control is not easy to increase the drug resistance of pathogenic bacteria, and has the advantage of environmental protection, so the biological control on the litchi anthracnose is strengthened, and the method gradually becomes the focus and hot spot of people, and has great significance for the development of the litchi industry.
Trichoderma spp fungus exists widely in soil and other base material, has the features of high survival capacity, wide adaptability, no environmental pollution, etc. and is one kind of broad spectrum antagonistic bacterium. The study of the use of Trichoderma to control plant diseases originated in 1932, and Weidling first discovered that Trichoderma lignicolum (Trichoderma lignorum) has a heavy parasitic effect on the hyphae of the plant pathogenic fungus Rhizoctonia solani (Rhizoctonia solani), and then many studies of the use of Trichoderma to biologically control plant diseases have been successively published. Zhang Wen et al found that Trichoderma atroviride T2 strain and its fermentation liquid all have strong inhibitory action on Trichothecium roseum (Trichoderma roseum), Fusarium graminearum (Fusarium latericum) and Alternaria mali (Alternaria mali), which are main pathogens of apple moldy core; the Xiaojie et al report that Trichoderma pseudokoningii TkonT1 strain has good bacteriostatic effect on Fusarium oxysporum, and the strain TkonT1 can be wound on the mycelium of the Fusarium oxysporum or penetrate into the mycelium to parasitize and grow, absorb the nutrients of the mycelium of the pathogen to cause the rupture of the mycelium of the pathogen and the digestion of the cell protoplasm; researches in Cao Cui Ling and the like find that Trichoderma koningii has strong antagonistic action on Helianthus annuus sclerotinia sclerotiorum. At present, some microbial agents containing trichoderma components are applied and have obvious effects, such as trichoderma harzianum T39 listed in Israel and trichoderma harzianum T-22 in America. Therefore, trichoderma pseudokoningii has attracted extensive attention as an important natural resource and has the potential of developing biopesticides.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art and develop application of Trichoderma pseudocorniculatum, the invention aims to provide Trichoderma pseudocorniculatum named Trichoderma pseudocorniculatum TJ5, which is separated by taking microorganisms on dry branches and fallen leaves of litchi as a screening object, has a remarkable inhibiting effect on anthracnose of litchi, provides new resources and directions for replacing chemical control by biological control, and can be developed and utilized as biological pesticide in the future.
The invention also aims to provide a biological agent prepared on the basis of the trichoderma pseudokoningii.
The invention further aims to provide application of the trichoderma pseudocorniculatum in preventing and treating litchi anthracnose.
The purpose of the invention is realized by the following scheme:
a strain of Trichoderma pseudokoningii named as Trichoderma koningiensis TJ5, deposited in Guangdong province culture Collection of microorganisms, GDMCC for short, address: the microbial research institute of Guangdong province, No. 59 building, No. 5 building, of Mieli Zhonglu, Guangzhou City, has a deposition number of GDMCC No. 61129, and a preservation time of 8 months and 8 days in 2020.
The trichoderma pseudocorniculatum is cultured on a PDA culture medium at 28 ℃, the growth speed is high, a bacterial colony grows over the whole culture dish after 3 days, hyphae are initially white and in a spider-shaped shape, a large amount of aerial hyphae exist, and the hyphae begin to form concentric rings and gradually turn green along with the extension of the culture time. The main branch of the conidiophores is longer, the secondary branch is nearly a right-angled branch, and the conidiophores are green, monospore, oblong or elliptical.
A biological agent is prepared based on the above Trichoderma pseudokoningii.
The biological agent is prepared by liquid fermentation of the trichoderma pseudokoningii, and preferably comprises the following steps: inoculating the trichoderma pseudokoningii to a PDB culture solution for culture to obtain the biological agent.
The pH value of the PDB culture solution is 7.0.
The culture refers to the culture for 5d under the conditions of 28 ℃ and shaking speed of a shaking table of 180 rpm.
The invention also provides application of the biological agent in preventing and treating litchi anthracnose.
The invention also provides application of the trichoderma pseudocorniculatum in preventing and treating litchi anthracnose.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the antagonistic bacteria screened from the litchi chinensis Sonn is mutually symbiotic with the litchi chinensis Sonn for a long time, so that a series of similar survival mechanisms are formed, the litchi chinensis Sonn is used for preventing and treating litchi chinensis diseases, and the litchi chinensis Sonn is safer and more reliable for eating litchi chinensis Sonn and processing products thereof.
(2) The trichoderma pseudocorniculatum has an excellent inhibitory effect on litchi anthracnose pathogen, has strong bacteriostatic ability, is environment-friendly and nontoxic in biological source, has small influence on the ecological environment, and has important practical significance for preventing and treating litchi anthracnose.
(3) The trichoderma pseudokoningii obtained by the method has the advantages of low requirement on culture conditions, strong viability and wide adaptability, and has good development and application prospects when being used as biocontrol bacteria.
Drawings
FIG. 1 is a single colony of Trichoderma pseudokoningii TJ5 on a PDA medium plate.
FIG. 2 shows conidiophores and conidiophores of Trichoderma pseudokoningii TJ 5. Wherein, A is a conidiophores diagram, and B is a conidiophores diagram.
FIG. 3 is a graph showing the inhibitory effect of Trichoderma pseudokoningii TJ5 on litchi anthrax. Wherein A is a colony morphology diagram of normal growth of litchi anthrax, and B is a diagram of inhibition effect of Trichoderma pseudocorninis on litchi anthrax.
FIG. 4 is a graph showing the inhibitory effect of a biological agent of Trichoderma pseudokoningii TJ5 on litchi anthrax. Wherein A is a colony morphology diagram of normal growth of litchi anthrax, and B is a diagram of inhibition effect of biological agent of Trichoderma pseudocorningii on litchi anthrax.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1: isolation, purification and preservation of Trichoderma pseudokoningii
(1) Preparing a PDA culture medium: firstly, cleaning and peeling potatoes, then weighing 200g of potatoes, cutting the potatoes, putting the potatoes into a pot, adding 800mL of water, boiling for 0.5h, filtering the mixture by eight layers of gauze, adding 20g of glucose, adding water to a constant volume of 1000mL, uniformly stirring the mixture by a glass rod, adding 15g of agar, fully heating and dissolving the agar, then subpackaging the mixture into a 250mL triangular flask, carrying out damp-heat sterilization at 121 ℃ for 20min, and preserving the mixture for later use.
(2) The trichoderma strain sample is separated from a green mildew layer on the dry branches and fallen leaves of litchi in a litchi base of garden institute of agriculture university in south China, and the separation and purification of the strain are carried out by a method of reference equation Zhongda and the like, and are slightly modified: picking a little green mould layer on a PDA culture medium plate by using an inoculating needle, and culturing in an incubator at 28 ℃ until the green mould layer is ready to be culturedAfter the green conidia grow out, pouring a proper amount of sterilized water to wash the conidia to prepare a conidia suspension, sucking 1mL of the conidia suspension, adding 9mL of sterile water, fully mixing, repeatedly diluting in the same way until the concentration of the conidia suspension is diluted to 10-4Then, 20. mu.L of the diluted spore suspension was pipetted and spread evenly on a plate containing PDA medium, and cultured at 28 ℃ for 2-3 days. Single colonies were picked and pure isolates of single spore cultures were obtained.
The isolated and purified strain was subjected to ITS sequence analysis, and was identified as Trichoderma pseudokoningii (Trichoderma koningiensis) and named Trichoderma pseudokoningii TJ 5. It has 100% homology with Trichoderma koningiensis isolate Tkois1 with accession number MT102394.1 in GenBank, and has been proved to be classified and named as Trichoderma pseudokoningiensis (Trichoderma koningiensis) and TJ 5. The sequence fragments are shown below:
CCTGCGGAGGGATCATTACCGAGTTTACAACTCCCAAACCCAATGTGAACCATACCAAACTGTTGCCTCGGCGGGGTCACGCCCCGGGTGCGTCGCAGCCCCGGAACCAGGCGCCCGCCGGAGGGACCAACCAAACTCTTTCTGTAGTCCCCTCGCGGACGTTATTTCTTACAGCTCTGAGCAAAAATTCAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCGGCGTTGGGGATCGGGAACCCCTAAGACGGGATCCCGGCCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGCGCAGTAGTTTGCACAACTCGCACCGGGAGCGCGGCGCGTCCACGTCCGTAAAACACCCAACTTTCTGAAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAAA。
the trichoderma pseudocorniculatum is cultured on a PDA culture medium at 28 ℃, the growth speed is high, the colony grows over the whole culture dish after 3d, the hyphae is initially white, is in a spider-line shape and has a large amount of aerial hyphae, and the hyphae begin to form concentric rings and gradually turn green along with the time extension (figure 1). The conidiophores have longer main branches and the secondary branches are nearly right-angled branches, and the conidiophores are green, monosporous, oblong or elliptical (figure 2).
(3) Deposit of trichoderma pseudokoningii TJ 5: the strain is preserved in Guangdong province microorganism culture collection center at 8 months and 8 days in 2020, GDMCC for short, and the address is as follows: the microbial research institute of Guangdong province, No. 59 building, No. 5 building, of Mieli Zhonglu, Guangzhou City, the number of which is GDMCC No. 61129.
Example 2: determination of trichoderma pseudokoningii activity by confrontation culture method
(1) PDA medium was prepared as in example 1.
(2) Preparation of single colony and bacterial dish of trichoderma pseudokoningii: picking trichoderma pseudokoningii hyphae with a sterilized toothpick on a PDA culture medium flat plate, and culturing for 3-4 days at 28 ℃ to obtain a single colony; and punching holes on the edges of the trichoderma pseudocorniculatum colonies by using a puncher to obtain the circular bacterium plate.
(3) Culturing litchi colletotrichum and preparing a bacterial dish: inoculating litchi anthrax strain stored at-20 deg.C to PDA culture medium plate for activation, and punching the edge of colony with a punch with diameter of 5mm after the colony grows over the plate to obtain circular bacterial dish.
(4) And (3) activity determination: and (3) inoculating the trichoderma pseudokoningii bacterial plate prepared in the step (2) and the litchi anthrax bacterial plate prepared in the step (3) to a PDA (personal digital assistant) plate culture medium of 9cm at a distance of 5cm respectively for opposite culture, taking singly inoculated litchi anthrax as a reference in an experiment, repeating the treatment for 3 times, culturing at a constant temperature of 25 ℃, observing the growth condition of bacterial colonies every day from the 2 nd, and measuring the growth diameter of the bacterial colonies after the bacterial colonies of a reference group grow to fill the culture dish (6 d). And (6) calculating the bacteriostasis rate. The result shows that the trichoderma pseudocorniculatum has obvious inhibition effect on colony growth of litchi anthrax, the inhibition rate reaches 70.73%, and the trichoderma pseudocorniculatum can completely or partially cover pathogenic fungi at the later stage and generate a large amount of conidia on the pathogenic fungi (figure 3).
Inhibition (%) - (control colony radius-treatment colony radius)/control colony radius x 100
Example 3: determination of activity of trichoderma pseudokoningii biological agent by growth rate method
(1) Preparation of PDA medium the same as in example 1; the preparation method of PDB liquid culture medium is the same as that of PDA solid culture medium except that agar is not added, the weighed medicine is fully dissolved and then is subpackaged into conical flasks (each flask is filled with 100mL of culture solution), the conical flasks are plugged and bound, moist heat sterilization is carried out at 121 ℃ for 20min, and the mixture is cooled and stored for later use.
(2) Preparation of a trichoderma pseudokoningii sterile fermentation filtrate (biological preparation): inoculating the trichoderma pseudokoningii bacterial plate prepared in the step (2) in the embodiment 2 into the conical flask filled with 100mL of PDB culture solution prepared in the step (1), placing the conical flask at 28 ℃, carrying out light-shielding oscillation culture for 5 days at the shaking table speed of 180rpm, centrifuging the conical flask at 5000rpm for 10min, sucking supernatant, and filtering out bacteria by using a filter with the aperture of 0.2 mu m to obtain the trichoderma pseudokoningii sterile filtrate.
(3) The procedure for culturing litchi colletotrichum and preparing the bacterial dish is the same as that of example 2.
(4) And (3) activity determination: and (3) mixing the biological preparation of the trichoderma pseudokoningii prepared in the step (2) and a PDA culture medium according to the ratio of 1: 9, and then pouring the plate, and pouring 15mL of the solution into each dish. The center of the plate is respectively connected with a trichoderma pseudokoningii bacterial plate and a litchi anthrax bacterial plate with the diameter of 5mm, the plates are placed at the temperature of 25 ℃ for constant-temperature culture until a control group grows full of the culture dish, and PDB is used for replacing a biological preparation of trichoderma pseudokoningii in the control group, and the treatment is repeated three times each time. And measuring the diameter of the experimental group after the control group bacterial colony grows over the culture dish, and calculating the bacteriostasis rate. The result shows that the biological agent of the trichoderma pseudocorniculatum TJ5 has an obvious inhibiting effect on litchi anthracnose pathogen, and the bacteriostasis rate is 27.5% (figure 4).
Inhibition (%) - (control colony radius-treatment colony radius)/control colony radius x 100
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.
Sequence listing
<110> southern China university of agriculture
<120> trichoderma pseudocorniculatum and application thereof in preventing and treating litchi anthracnose
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 583
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ITS sequence of Trichoderma pseudokoningii (Trichoderma koningiopsis) TJ5
<400> 1
cctgcggagg gatcattacc gagtttacaa ctcccaaacc caatgtgaac cataccaaac 60
tgttgcctcg gcggggtcac gccccgggtg cgtcgcagcc ccggaaccag gcgcccgccg 120
gagggaccaa ccaaactctt tctgtagtcc cctcgcggac gttatttctt acagctctga 180
gcaaaaattc aaaatgaatc aaaactttca acaacggatc tcttggttct ggcatcgatg 240
aagaacgcag cgaaatgcga taagtaatgt gaattgcaga attcagtgaa tcatcgaatc 300
tttgaacgca cattgcgccc gccagtattc tggcgggcat gcctgtccga gcgtcatttc 360
aaccctcgaa cccctccggg gggtcggcgt tggggatcgg gaacccctaa gacgggatcc 420
cggccccgaa atacagtggc ggtctcgccg cagcctctcc tgcgcagtag tttgcacaac 480
tcgcaccggg agcgcggcgc gtccacgtcc gtaaaacacc caactttctg aaatgttgac 540
ctcggatcag gtaggaatac ccgctgaact taagcatatc aaa 583
Claims (6)
1. Trichoderma pseudokoningii (f)Trichoderma koningiopsis) TJ5, deposited in the Guangdong province culture Collection of microorganisms, GDMCC for short, address: the microbial research institute of Guangdong province, No. 59 building, No. 5 building, of Mieli Zhonglu, Guangzhou City, has a deposition number of GDMCC No. 61129, and a preservation time of 8 months and 8 days in 2020.
2. A biological agent, which is characterized by being prepared by a method comprising the following steps: inoculating Trichoderma pseudokoningii TJ5 as defined in claim 1 into PDB culture solution, and culturing to obtain biological preparation.
3. The biological agent according to claim 2, characterized in that the PDB broth has a pH of 7.0.
4. The biological agent according to claim 2, wherein the incubation is carried out at 28 ℃ and shaking table at a shaking speed of 180rpm for 5 days.
5. Use of a biological agent according to any one of claims 2 to 4 for the control of litchi anthracnose.
6. The use of trichoderma pseudocorniculatum TJ5 of claim 1 for the control of litchi anthracnose.
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