CN116024094A - Cyst-philic basket fungus GX1 and application thereof - Google Patents
Cyst-philic basket fungus GX1 and application thereof Download PDFInfo
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Abstract
The invention discloses a strain of bastardtoader GX1, which is bastardtoader (Talaromyces cystophila) GX1 with a preservation number of CGMCC No.40002. The metabolite of the cyst-basket bacteria GX1 is applied to the biological control of plant parasitic nematodes such as southern root-knot nematodes, root-knot nematodes of like-earbeans, soybean cyst nematodes, corn cyst nematodes, root-rot nematodes and the like. The metabolite of the cyst-basket bacterial strain has obvious nematicidal active substances, can be used for biological control of plant parasitic nematodes such as meloidogyne incognita, soybean cyst nematodes, corn cyst nematodes, root rot nematodes and the like, and has the potential of being developed as a nematicide.
Description
Technical Field
The invention belongs to the technical field of microbial pesticides, and particularly relates to a strain of basket-shaped sporophore GX1 and application thereof.
Background
Plant parasitic nematodes cause serious diseases of crops, and are difficult to control due to the concealment of the plant parasitic nematodes, and meanwhile, chemical nematicides with high toxicity and high residues are very harmful to human and animal health and environment, so that an alternative method for chemical control is needed to be searched for in production. Among them, the development of microorganisms and their metabolites has been a research hotspot in recent years. There are many kinds of microbial control resources, mainly including: natural enemy fungi, natural enemy bacteria, natural enemy actinomycetes, and other nematode natural enemy organisms, and the like. The natural enemy fungi of the nematodes are widely distributed, and many reports are already made in the study of biological control of plant parasitic nematodes. There are 700 species of nematophagous fungi reported worldwide, including 380 species of nematophagous fungi, 120 species of parasitic fungi in nematodes, 270 species of toxigenic fungi and a large number of opportunistic fungi (Zhang Ying et al, 2011). For abundant nematophagous fungi resources, scientists around the world have conducted extensive research in recent years. Verticillium chlamydia (also known as Podoconia thick Pochonia chlamydosporium), paecilomyces lilacinus (Paecilomyces lilacinus), verticillium lecanii (Verticillium lecanii), mortierella (Hirsutella rhossiliensis, H.minnesotensis), fusarium (Fusarium), and the like. There is also an increasing research on the development of nematicidal active substances from microbial metabolites, and at present, approximately 230 metabolites active on nematodes have been isolated from more than 150 strains of genus 280, mainly including quinones, alkynes, sterols, peptides, heterocycles, ceramides, lignans, macrolides, fatty acids, presomeerins, piperazines, peptides, furans, etc. produced from part of the bacteria in fungi (Deuteromycetes), basidiomycetes (Basidiomycetes), ascomycetes (Ascomycetes). Trichoderma (Trichoderma) is one of the most studied effective biocontrol fungi worldwide and plays an important role in agricultural production, and it can not only parasitize eggs and larvae, but also produce a large number of secondary metabolites with nematicidal activity (Li et al 2015). Acremonium sp BH0531 has been shown to be a marine fungus with good control effects on root-knot nematodes, and 40mL of BH0531 fermentation broth with mass concentrations of 5, 10, 20 and 40mg/mL is added to soil, so that damage caused by root-knot nematodes to cucumber plants can be effectively relieved, and plant resistance is enhanced (Cai Shuang and the like, 2016).
Disclosure of Invention
The invention aims to provide a soil microorganism with a cyst-basket strain and subtropical climate conditions, which has good parasitism on cyst nematodes, and the metabolite of the soil microorganism has good poisoning effect on cyst nematodes and root knot nematode second-instar larvae, has high virulence biocontrol strain on plant parasitic nematodes, has parasitic effect on cyst nematodes and good effect of poisoning second-instar larvae, and has very important significance on enriching plant parasitic nematode biocontrol bacterial resources and developing nematicide microorganism source pesticides or pesticide fertilizers.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
the strain of the bastard fungus GX1 is named as bastard fungus (Talaromyces cystophila) GX1, the preservation number is CGMCC No.40002, the preservation date is 2021, 12 months and 06 days, the preservation unit is China general microbiological culture Collection center, and the preservation address is North Xicilu No. 1, 3 in the Korean region of Beijing city.
The application of the cyst-forming fungus GX1 in the biological control of plant parasitic nematodes such as southern root-knot nematodes, root-knot nematodes of like-earbeans, soybean cyst nematodes, corn cyst nematodes, root-rot nematodes and the like.
The application of the metabolite of the cyst-philic basket fungus GX1 in the biological control of plant parasitic nematodes such as southern root-knot nematodes, root-knot nematodes of like-bean, soybean cyst nematodes, corn cyst nematodes, root-rot nematodes and the like.
Preferably, the preparation method of the metabolite of the sporobasket bacteria GX1 comprises the steps of culturing the sporobasket bacteria Talaromyces cystophila GX1 on a PDA flat plate for 5-7 days, then beating the bacterial colony obtained by culture into bacterial cakes, inoculating the bacterial cakes into Charles culture solution, respectively carrying out shaking culture for 1-2 weeks at the inoculum size of 1 bacterial cake at the temperature of 26 ℃ and 150r/min, and filtering the obtained liquid, namely the metabolite of the sporobasket bacteria Talaromyces cystophila GX1.
Preferably, the metabolite of the cyst-philic basket bacteria GX1 is prepared into a preparation for preventing and controlling the biological control of plant parasitic nematodes such as southern root-knot nematodes, meloidogyne incognita, soybean cyst nematodes, corn cyst nematodes and root rot nematodes.
Compared with the prior art, the invention has the following beneficial technical effects:
the metabolite of the cyst-basket bacterial strain has obvious nematicidal active substances, can be used for biological control of plant parasitic nematodes such as meloidogyne incognita, soybean cyst nematodes, corn cyst nematodes, root rot nematodes and the like, and has the potential of being developed as a nematicide.
Description of preservation information
The bastardtoadenomyces sporophore (Talaromyces cystophila) GX1 is preserved in China general microbiological culture Collection center (CGMCC) for 12 and 06 days in 2021, and the preservation number is CGMCC No.40002.
Drawings
FIG. 1 shows the morphology of the basket (Talaromyces cystophila) GX1 strain of the present invention; wherein A is a colony on a CYA medium; b is a colony on the MEA culture medium; c is a colony on YES medium; d is a colony on the OA medium; e is a colony on PDA medium.
Detailed Description
The following detailed description, in conjunction with the accompanying drawings, describes in detail, but it is to be understood that the scope of the invention is not limited to the specific embodiments. The raw materials and reagents used in the examples were commercially available unless otherwise specified. The experimental methods used in the following examples are conventional methods unless otherwise specified. The quantitative experiments in the following examples were all performed in triplicate, and the results were averaged.
The composition of the medium used in the examples is as follows:
20% Charles medium: naNO 3 2.00g，KCl 0.50g，FeSO 4 0.01g，K 2 HPO 4 1.00g，MgSO 4 0.50g of sucrose 30.0g is dissolved in 1000ml of distilled water, and is stirred continuously by a glass rod, and is split into 10 triangular flasks of 250ml after being fully dissolved, and is sterilized by high-pressure steam (121 ℃) for 20 minutes for standby;
CYA medium: naNO 3 3.0g，KH 2 PO 4 1.0g，KCl 0.5g，MgSO 4 ·7H 2 O 0.5g，FeSO 4 ·7H 2 0.01g of O, 5.0g of yeast extract, 30.0g of sucrose, 15.0g of agar and 1000ml of distilled water;
yeast extract 20g, sucrose 150g, magnesium sulfate heptahydrate 0.5g, traces of salt solution (FeSO 4 .7H 2 O 0.1g MnCl 2 .4H 2 O0.1 g), agar 20g, distilled water 885ml, and the mixture was thoroughly mixed and autoclaved at 121℃for 15min at pH 6.5.+ -. 0.2.
MEA malt extract (Oxoid CM 0059) 50g, a trace amount of salt solution 1ml, distilled water 1000ml, and sterilizing at 121deg.C for 10min with pH 5.4+ -0.2.
OA, oat flour infusion 30g, traces of saline solution 1ml, agar 20g, distilled water 1000ml, pH 6.5.+ -. 0.2.
PDA potato dextrose agar medium.
Example 1
Sample collection and strain separation and purification
The soil sample is collected from corn lands in Xinbing district of Guangxi guest city, Z-shaped sampling method is adopted to collect the soil sample, 1-2cm surface soil is removed from the root of the plant, and the soil sample is taken; separating the cyst of the nematode in the soil by adopting a elutriation sieving method, transferring the mycelium cyst growing out into a PDA flat plate for culture by adopting a filter paper sheet moisturizing culture method, further separating and purifying to obtain different strains, and respectively carrying out activity verification such as parasitic capacity and the like to obtain the strain GX1 with high parasitic rate.
Morphological identification of strains
Strain GX1 was inoculated onto CYA, YES, MEA, OA and PDA, respectively, and after culturing at 25 ℃ for 7d, the characteristics of the colony such as color, texture, spore production degree and pigment secretion were observed and recorded, and morphological characteristics of the strain were microscopically observed and recorded (fig. 1).
Molecular biology identification of strains
The genome DNA of the strain is extracted according to the instruction of a rapid extraction kit of the genome DNA of the biological fungi, and the extracted DNA is preserved in a refrigerator at the temperature of minus 20 ℃. Strains Internal Transcribed Spacer (ITS), beta-tubulin (BenA), calmodulin (CaM) and RNA polymerase II second largest subunit (RPB 2) genes were amplified, amplification primers TCCGTAGGTGAACCTGCGG (5 '-3'), TCCTCCGCTTATTGATATGC (3 '-5'), and amplified products showed a single, bright amplified segment length band (ITS of about 600bp, benA of about 500bp, caM of about 700bp, RPB2 of about 1000 bp) and sequenced. The amplification parameters are ITS:94 ℃,5min,94 ℃,45s,55 ℃,45s,72 ℃,1min, (35 cycles) 72 ℃, for 7min; caM:94 ℃,5min,94 ℃,45s,55 ℃,45s,72 ℃,1min, (35 cycles) 72 ℃, for 7min; benA:94 ℃,5min,94 ℃,30s,55 ℃,45s,72 ℃,1min, (35 cycles) 72 ℃,7min; RPB2:94 ℃,5min,94 ℃,45s,60 ℃,45s,72 ℃,1min, (35 cycles) 72 ℃,7min. Sequencing the amplified genes to obtain the sequences of the genes.
Phylogenetic analysis
The original sequence is manually checked and edited by biological software Bioedit 7.0.9, accurate full-segment sequences are obtained by MEGA 7.0 software and then submitted to GenBank, a model strain reported by the genus Brucella is used as a reference, single gene sequences are spliced into a polygenic sequence combination of ITS-BenA-CaM-RPB2, cluster analysis is carried out by MEGA 7.0, and the strain is identified as the basophilic basket Talaromyces cystophila by morphological and molecular biological methods and is named Talaromyces cystophila GX1.
Example 2
Preparation of metabolite of basket-shaped sporophore Talaromyces cystophila GX1
The cyst-forming bacteria Talaromyces cystophila GX1 are cultured on a PDA flat plate for 5-7 days, bacterial colonies obtained by culture are inoculated into a triangular flask (250 mL) filled with 100mL of Charles culture solution by using a puncher (diameter is 0.9 mm), the inoculation amount is 1 bacterial cake, the bacterial colonies are respectively cultured for 1 week, 2 weeks and 3 weeks in a shaking way at 26 ℃ and 150r/min, and the liquid obtained by filtering with a 0.22 mu m bacterial filter is fermentation filtrate stock solution, namely a metabolite of the cyst-forming bacteria Talaromyces cystophila GX1 for later use and is used for nematicidal tests.
Example 3
Parasitic action of basket-like cyst Talaromyces cystophila GX1 on cysts
The cyst-forming basket bacteria Talaromyces cystophilaGX1 are cultured by a PDA plate at the temperature of 25 ℃, and the surface-sterilized cysts are connected at the edge of the colony when the radius of the colony in the plate is as large as 1/2 of the radius of a culture dish. 10 cysts were randomly picked out every 2 days, the surface of 70% alcohol was sterilized for 1min, then the mixture was returned to the PDA plate, the mixture was repeated 3 times, and the cyst parasitism was recorded, and the cyst parasitism rate of the strain GX1 was counted and represented by the ratio of the parasitic rate= (the number of the cysts of the mistletoe per total number of the cysts after the return) ×100%, and the results were shown in Table 1 below.
Results: parasitism of bursaphelenchus xylophilus cyst by bursaphelenchus xylophilus Talaromyces cystophila GX1
The study result shows that after the strain GX1 is treated, the cysts are disinfected on the surface and are returned to the PDA, the central dark green edge light green colony is regrown, and after the strain GX1 is treated on the cysts for 3d, the average parasitism rate of three experiments is 91.1 percent.
Table 1 parasitism of bursa of corn cyst nematode after 3 days of strain GX1 (%)
Example 4
Corn cyst line egg and 2-year larva collection
Preparation of nematode egg suspension: selecting full white cysts, sterilizing the surface of the cysts with 70% alcohol for 3min, fully flushing with sterile water for 3-5 times, lightly crushing the cysts with rubber plugs to release eggs, pouring the egg suspension into 200-mesh and 500-mesh screens, flushing with sterile water for several times, and collecting the egg suspension to obtain nematode egg suspension for later use.
Preparation of a suspension of second instar larvae (J2): hatching the surface sterilized cysts under the dark condition of 33 ℃, and collecting the hatched second-instar larvae on the test day to obtain the nematode suspension.
Research on influence of metabolite of cyst basket Talaromyces cystophila GX1 on hatching and ovicidal effects of corn cyst nematode eggs
The stock solutions of the fermentation filtrates prepared in example 2 (metabolite of the bursaphelenchus xylophilus Talaromyces cystophila GX) were mixed with sterile water to prepare fermentation filtrates having volume concentrations of 2.5%, 5%, 10%, 20%, 50%, respectively, 200. Mu.L of the fermentation filtrates having concentrations of 2.5%, 5%, 10%, 20%, 50% were aspirated for 1 week, 2 weeks and 3 weeks, respectively (i.e., filtrates having concentrations of 2.5%, 5%, 10%, 20%, 50% were aspirated for 1 week, 200. Mu.L of the filtrates having concentrations of 2.5%, 5%, 10%, 20%, 50% respectively, were aspirated for 2 weeks, and so on), and then added to a sterile 96-well cell culture plate, followed by adding to each well about 60 of the above-mentioned maize cyst line eggs in suspension for use. Each treatment concentration was repeated 4 times with 20% chalcogen medium and sterile water as controls. The 96-well cell culture plates were placed in a constant temperature incubator at 33℃without light, then the incubation of eggs was observed under a Ti-S Nikon microscope at 3d, 6d, 9d, 12d and 15d, respectively, and the number of hatched corn cyst nematodes J2 was recorded and the test was repeated 3 times. Hatchability= (number of hatched J2/number of test eggs) ×100% and the results are shown in table 2 below.
Results: the metabolite of the aschersonia aleyrodis Talaromyces cystophila GX1 has remarkable ovicidal effect on the corn cyst nematodes
The research results show that: the concentration of 2 weeks fermentation broth (metabolite of cyst-forming basket Talaromyces cystophila GX1 prepared in example 2) was 2.5%, 5%, 10%, 20%, 50% of the fermentation filtrate, the cumulative hatchability of the corn cyst wire eggs after 15d treatment was 33.94%, 19.03%, 6.96%, 2.17%, 2.53%, significantly lower by 20% of the chalcogen medium and sterile water control treatment (P < 0.05) (table 2), and the non-hatched eggs all formed vacuolated deaths, losing hatching ability.
TABLE 2 influence of 2 week fermentation filtrate of GX1 Strain on the J2 ratio (%) of hatching of corn cyst nematode eggs
Note that: data are mean ± standard error. The different letters following the same column of values represent significant differences at levels P <0.05 according to the LSD method test.
Toxic action of metabolite of basket-shaped cyst Talaromyces cystophila GX1 on two-stage cyst nematode larvae
200. Mu.L of the fermentation filtrate stock (metabolite of Saprolegnia sporogenes Talaromyces cystophila GX1 as prepared in example 2) prepared in example 2 was pipetted into a sterile 96-well cell culture plate at a concentration of 2.5% (volume of fermentation broth: volume of sterile water=1:39), 5%, 10%, 20%, 50% for 1 week, 2 weeks of fermentation, and J2 incubated on the same day (suspension of two-age larvae (J2) prepared as described above) was added to the 96-well cell culture plate, about 60 per well. Each treatment concentration was repeated 4 times with 20% chalcogen medium and sterile water as controls. The 96-well cell culture plate is placed in a constant temperature incubator without illumination at 25 ℃, and then the death condition of nematodes in each treatment is detected by a microscope for 24, 48 and 72 hours respectively, and the judgment standard of the death of the nematodes is that: nematode stiffness and inactivity following needle stimulation, indicating nematode death (Choi et al, 2007), mortality and corrected mortality of nematodes were calculated and the test repeated 3 times.
Mortality = (number of dead nematodes/number of nematodes tested) ×100%
Results: metabolite of bursaphelenchus xylophilus Talaromyces cystophila GX1 for poisoning second-instar larvae of corn cyst nematodes
The results showed that the mortality rate of the corn cyst nematodes J2 treated with the filtrates at different concentrations showed a significant difference at 24h in the fermentation filtrate (metabolite of the cyst basket Talaromyces cystophila GX1 prepared in example 2) fermented for 1 week, the mortality rate was 2.5% at the lowest, 10.23% at the lowest and 83.74% at the highest. After 72h, 10%, 20% and 50% of the fermentation filtrate is used for treating the corn cyst nematode J2, and the death rate is 100%. In the fermentation filtrate of 2 weeks, 50% of the fermentation filtrate is used for treating the corn cyst nematode J2, the death rate reaches 100%, and 5%, 10% and 20% of the fermentation filtrate is used for treating the corn cyst nematode J2, so that the death rate is higher than 90%. Significantly higher than the control group (P < 0.05) (table 3).
TABLE 3 mortality of the treatment of corn cyst nematode J2 with various fermentation time concentrations Talaromyces cystophila GX1 Strain fermentation filtrate%
Note that: data are mean ± standard error. The different letters following the same column of values represent significant differences at levels P <0.05 according to the LSD method test.
Podophyllum sporophore Talaromyces cystophila GX1 metabolite poisoning effect on two-instar larvae of root knot nematode
The specific study and calculation method were the same as above, except that the treatment of fermentation filtrate stock solutions (metabolite of the standby bursa-pastoris Talaromyces cystophila GX1 of example 2) for 1 week, 2 weeks and 3 weeks of fermentation was used in the present test.
Results: the Talaromyces cystophila GX strain fermentation liquor has obvious poisoning effect on the second-instar larvae of root-knot nematodes (like the root-knot nematodes of the auricularia auricula): the experimental procedure was as in example 3.
After the strain is treated by fermentation liquor for 24 hours, all nematode bodies are stiff, cavitation appears, and the death rate reaches 100%. Most of the nematodes are stiff after 24 hours of treatment by the fermentation liquid for 2 weeks, cavitation appears in the worms after 48 hours, and all the nematodes die after 72 hours (table 4), so that the insecticidal effect is very good.
Table 4 Talaromyces cystophila GX1 mortality of the strain at different times after treatment of the group-knot nematode J2 of Earbean at different fermentation times (%)
Note that: each treatment was repeated 4 times with about 50J 2 replicates each. Different letters from the same column represent a significant difference at the 0.05 level (LSD method).
The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (5)
1. The strain of the bastardtoadenomyces lanuginosus GX1 is characterized in that the collection number of the bastardtoadenomyces lanuginosus (Talaromyces cystophila) GX1 is CGMCC No.40002.
2. Use of the cyst-philic basket fungus GX1 according to claim 1 for biological control of meloidogyne incognita, soybean cyst nematode, corn cyst nematode, and plant parasitic nematode of meloidogyne incognita.
3. Use of a metabolite of the basket-like cyst-forming fungus GX1 according to claim 1 for the biological control of root-knot nematodes in the south, root-knot nematodes in the weevil, soybean cyst nematodes, corn cyst nematodes, root rot nematodes and plant parasitic nematodes.
4. A use according to claim 3, characterized in that: the preparation method of the metabolite of the sporobasket bacteria GX1 comprises the steps of culturing the sporobasket bacteria Talaromyces cystophila GX on a PDA flat plate for 5-7 days, then beating the bacterial colony obtained by culture into bacterial cakes, inoculating the bacterial cakes into Charles culture solution, respectively carrying out shaking culture for 1-2 weeks at 26 ℃ and 150r/min, and filtering the obtained liquid, namely the metabolite of the sporobasket bacteria Talaromyces cystophila GX1.
5. Use according to claim 3 or 4, characterized in that: the metabolite of the cyst-basket bacteria GX1 is prepared into a preparation and is applied to biological control of southern root-knot nematodes, like ear bean root-knot nematodes, soybean cyst nematodes, corn cyst nematodes and root-rot nematodes.
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| US20070259783A1 (en) * | 2003-10-29 | 2007-11-08 | Hideaki Tateishi | Fungus Having Activity of Controlling Disease of Gramineous Plant, Controlling Agent Using the Same, Method of Controlling and Biological Material |
| CN102286383A (en) * | 2011-09-06 | 2011-12-21 | 青岛农业大学 | Talaromyces flavus and application of same in prevention of plant pathogens |
| CN113151001A (en) * | 2021-02-02 | 2021-07-23 | 华中农业大学 | Talaromyces flavus strain TF-04 and application thereof |
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| US20070259783A1 (en) * | 2003-10-29 | 2007-11-08 | Hideaki Tateishi | Fungus Having Activity of Controlling Disease of Gramineous Plant, Controlling Agent Using the Same, Method of Controlling and Biological Material |
| CN102286383A (en) * | 2011-09-06 | 2011-12-21 | 青岛农业大学 | Talaromyces flavus and application of same in prevention of plant pathogens |
| CN113151001A (en) * | 2021-02-02 | 2021-07-23 | 华中农业大学 | Talaromyces flavus strain TF-04 and application thereof |
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| 金娜;刘倩;简恒;: "植物寄生线虫生物防治研究新进展", 中国生物防治学报, no. 05, 8 October 2015 (2015-10-08), pages 789 - 800 * |
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