CN114921346B - Rhizopus dysmorphicus APS-1 and application thereof - Google Patents
Rhizopus dysmorphicus APS-1 and application thereof Download PDFInfo
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- 241000235527 Rhizopus Species 0.000 title claims abstract description 23
- 241001553178 Arachis glabrata Species 0.000 claims abstract description 71
- 235000020232 peanut Nutrition 0.000 claims abstract description 70
- 235000017060 Arachis glabrata Nutrition 0.000 claims abstract description 55
- 235000010777 Arachis hypogaea Nutrition 0.000 claims abstract description 55
- 235000018262 Arachis monticola Nutrition 0.000 claims abstract description 55
- 238000004321 preservation Methods 0.000 claims abstract description 7
- 230000001737 promoting effect Effects 0.000 claims abstract description 7
- 241000303962 Rhizopus delemar Species 0.000 claims abstract description 4
- 239000000725 suspension Substances 0.000 claims description 9
- 241001663183 Rhizophagus irregularis Species 0.000 claims description 5
- 238000009629 microbiological culture Methods 0.000 claims description 4
- 239000002068 microbial inoculum Substances 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 2
- 201000010099 disease Diseases 0.000 abstract description 14
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 14
- 244000053095 fungal pathogen Species 0.000 abstract description 8
- 238000000034 method Methods 0.000 abstract description 6
- 230000002265 prevention Effects 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 244000005700 microbiome Species 0.000 abstract description 2
- 210000004215 spore Anatomy 0.000 description 17
- 241000196324 Embryophyta Species 0.000 description 11
- 230000000694 effects Effects 0.000 description 9
- 239000002689 soil Substances 0.000 description 8
- 241000233866 Fungi Species 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
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- 241001530056 Athelia rolfsii Species 0.000 description 2
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- 239000003899 bactericide agent Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
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- 238000011835 investigation Methods 0.000 description 2
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108020004463 18S ribosomal RNA Proteins 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 241000589156 Agrobacterium rhizogenes Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 241000233667 Saprolegnia Species 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, 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
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, 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
- C12N1/14—Fungi; Culture media therefor
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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Abstract
The invention belongs to the technical field of microorganisms, and relates to a strain of rhizopus delemar APS-1 and application thereof, wherein the strain has a preservation number of CGMCC No.40116. The strain APS-1 is applied to promoting peanut growth and preventing and controlling peanut pathogenic fungi, and the strain APS-1 is inoculated in the peanut growth process to promote peanut growth and improve the resistance of the peanut to the pathogenic fungi such as southern blight and the like. The invention relates to a method for promoting peanut growth and preventing and controlling peanut pathogenic fungi by using the special-shaped rhizopus APS-1, which belongs to the field of biological prevention and control, is safe, quick and effective, and is beneficial to healthy green production of peanuts and comprehensive prevention and control of peanut diseases.
Description
Technical Field
The invention belongs to the technical field of microorganisms, relates to a symbiotic fungus rhizopus radiatus, and in particular relates to separation and identification of a rhizopus dysmorphis APS-1 with a growth promoting effect on various peanut varieties and application of the rhizopus dysmorphis APS-1 in promoting peanut growth and preventing and controlling peanut pathogenic fungi.
Background
The peanuts are used as important cash crops and oil crops in China, the planting area is continuously increased, the economic benefit is good, but diseases seriously affect the healthy production of the peanuts, so that serious economic loss is caused, wherein the southern blight caused by the sclerotium rolfsii (Sclerotium rolfsii) is used as one of the most serious soil-borne diseases, and the yield loss of the peanuts is more than 50%. At present, chemical bactericides are commonly used for preventing and treating southern blight by adopting agricultural measures such as rotation with non-hosts or deep ploughing to cover infected crops, but the bactericides can cause the problems of pesticide residues, enhanced pathogen resistance, environmental pollution and the like, and the agricultural measures can only prevent diseases, so that a method for effectively controlling the southern blight and protecting the environment is needed to be found.
Biological control is a safe and environment-friendly substitute for crops, wherein arbuscular mycorrhizal fungi (Arbuscular mycorrhizalfungi, AMF) are a type of microbial resource with great application potential in the aspects of biological control of plant diseases and improvement of plant resistance/disease tolerance. AMF is a common resident in soil, can be symbiotic with more than 90% of terrestrial plants, and is a microbial resource with great application potential in the aspects of biological prevention and control of soil-borne diseases and improvement of plant resistance/disease resistance. The invention researches the control effect on peanut southern blight by establishing a symbiotic system of arbuscular mycorrhizal fungi and peanuts and by means of a reciprocal symbiotic relationship.
Disclosure of Invention
The invention aims to solve the problems in the prior art, and provides a strain of special-shaped rhizopus APS-1 and application thereof, wherein special-shaped rhizopus is inoculated on peanuts, and the symbiotic system of the special-shaped rhizopus APS-1 and the peanuts is established, so that the growth of the peanuts is promoted by means of a reciprocal symbiotic relationship, and the resistance of the peanuts to southern blight is improved.
The technical scheme of the invention is as follows:
The strain of the Saprolegnia heteromorphic (Rhizophagus irregularis) APS-1 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.40116.
The invention also protects the application of the rhizopus deleteri APS-1 in preventing and controlling peanut pathogenic fungi.
Further, the peanut pathogenic fungi include peanut southern blight.
Further, the application form is a spore suspension of strain APS-1.
The invention also protects the application of the rhizopus deleteri APS-1 in promoting the growth of peanuts.
Further, the application form is a spore suspension of strain APS-1.
The invention also provides a microbial inoculum for preventing and treating peanut southern blight, and the active ingredient of the microbial inoculum is the rhizopus delemar APS-1.
Biological material sample preservation information:
the strain APS-1 is the oocyst mould (Rhizophagus irregularis) which is preserved in China general microbiological culture Collection center (CGMCC), and the preservation address is: the collection date of the institute of microbiology, national academy of sciences, china, at the area North Star West way 1, 3, chaoyang, beijing: and the preservation number is CGMCC No.40116 in 22 days of 2022 and 03.
The invention has the beneficial effects that:
According to the invention, the special-shaped rhizopus APS-1 is inoculated in the peanut growth process, so that on one hand, the special-shaped rhizopus APS-1 can promote the absorption of mineral elements by the peanut, promote the peanut growth and improve the resistance of the peanut to biotic stress and abiotic stress through establishing a good symbiotic relation with the peanut; on the other hand, the rhizopus deleteri APS-1 can improve the resistance of peanuts to pathogenic fungi such as southern blight and the like.
The method for promoting the growth of the peanut and preventing and controlling the pathogenic fungi of the peanut by using the special-shaped rhizopus APS-1 belongs to the field of biological prevention and control, is safe, quick and effective, is beneficial to healthy and green production of the peanut, and provides technical support for comprehensive prevention and control of peanut diseases.
Drawings
FIG. 1 is a schematic diagram of co-cultivation of strain APS-1 with hairy roots of peanut;
FIG. 2 is a morphological feature of strain APS-1;
FIG. 3 is a phylogenetic tree of strain APS-1 constructed based on the SSU-ITS-LSU fragment;
FIG. 4 is a schematic microscopic view of the root system characteristics of peanut without inoculating and inoculating strain APS-1;
FIG. 5 shows the effect of strain APS-1 on peanut growth;
FIG. 6 is a schematic diagram of the calculation result of the dependence of peanut on mycorrhiza;
FIG. 7 shows the effect of strain APS-1 on the occurrence of peanut southern blight;
FIG. 8 shows the effect of strain APS-1 on controlling peanut southern blight.
Detailed Description
For a further understanding of the present invention, reference will now be made in detail to the present embodiments of the invention, examples of which are illustrated in the accompanying drawings. 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.
Unless otherwise indicated, materials, reagents and the like used in the following examples are all commercially available; unless otherwise specified, the experimental methods used in the examples below were all conventional.
Example 1
Separation, rapid propagation and identification of special-shaped rhizopus APS-1
1. Isolation of arbuscular mycorrhizal fungi:
Collecting peanut rhizosphere soil in Laixi city, taking soil 5-20 cm away from the ground surface, naturally airing in a shade, and separating arbuscular mycorrhizal fungi spores in the soil by a wet sieve decantation method. Spores were initially classified by means of a microscope, with the dominant bacterium designated APS-1. The strain APS-1 spores are spherical, nearly spherical or irregularly shaped, brown, have a long axis of 70-153 mu m and contain oily substances in the spores.
2. Rapid propagation of peanut hairy roots APS-1:
Culturing cotyledons of peanut variety flower-culturing No. 45 under aseptic condition, infecting peanut cotyledons with agrobacterium rhizogenes A4, inducing generation of transgenic peanut hairy roots, and obtaining a large number of hairy roots through screening and propagation. The spores of APS-1 obtained by separation are placed on MSR culture medium together with hairy roots of peanut after surface sterilization, and are dark-cultured at 25 ℃. When hypha and spore appear on the culture medium, the peanut root is subcultured to obtain peanut hairy root containing APS-1, as shown in figure 1, and then the mycorrhiza is cultured by double aseptic culture method to obtain a large amount of spores and hypha of APS-1, wherein the spore and hypha form is shown in figure 2.
3. Identification of strains:
Spores and mycelia of arbuscular mycorrhizal fungi APS-1 were collected, genomes were extracted, and SSU-ITS-LSU fragments were amplified by PCR using 18S rDNA universal primer SSU-Glom (5'ATTACGTCCCTGCCCTTTGTACA 3') (SEQ ID NO: 1) and 28S rDNA universal primer NDL22 (5'TGGTCCGTGTTTCAAGACG 3') (SEQ ID NO: 2), respectively. The PCR products were sequenced by Beijing engine biotechnology Co. The full length of the 28S rDNA sequence is 1377bp, and the sequence is shown as SEQ ID NO: 3.
The sequences obtained were aligned for BLAST analysis and the SSU-ITS-LSU fragments were used to construct phylogenetic trees using MEGA7 software, as shown in FIG. 3. The results show that strain APS-1 is on the same branch as Rhizopus arbuscular mycorrhizal fungi Rhizopus prinus Rhizophagus irregularis MUCL43205, indicating that strain APS-1 is Rhizopus prinus. The strain APS-1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 40116.
Example 2
Effect of strain APS-1 spore suspension on peanut growth
Spores and hyphae of strain APS-1 were collected and prepared as a spore suspension for use. The pots were filled with moist field soil, inoculated with a spore suspension of about 900 strains of APS-1 at 2/3 of the height of the pot, after which the pots were filled with soil and an equal volume of water was inoculated as a control.
Peanut variety Luhua No. 8 (LH 8), zhonghua No. 24 (ZH 24), baisha 1016 (BS 1016), yuhua 9620 (YH 9620), huayu 917 (HY 917) and Huayu 33 (HY 33) are sowed in the flowerpot, 4 seeds are sowed in each pot, and 3 peanuts are reserved after emergence. The control and treatment were repeated 3 times for 6 pots each, all peanuts were placed in a plastic greenhouse and watered periodically. 3 pots were taken after 4 weeks, the root systems were collected for trypan blue staining, and the infection of the root systems was observed by a microscope. After 8 weeks of peanut culture, the total fresh weight of the plants is weighed, and the total dry weight of the plants is weighed after drying. Mycorrhizal dependency is calculated as follows: mycorrhiza dependency (%) = (inoculation dry weight-non-inoculation dry weight)/inoculation dry weight x 100. The microscopic examination result of the peanut root system is shown in fig. 4, wherein fig. 4A is the characteristics of the peanut root system which is not inoculated, and fig. 4B and fig. 4C are the characteristics of the peanut root system of the inoculated strain APS-1; the microscopic examination of peanut root system finds that 6 peanuts can be symbiotic with the strain APS-1.
As shown in fig. 5 (in the figure, the difference is very significant at the p <0.01 level), it was found that strain APS-1 was able to significantly increase the main stem length, side branch length, total fresh weight and total dry mass of 6 peanut varieties compared to the control.
As can be seen from fig. 5, the main stem lengths of the luhua No. 8, the medium flower No. 24, the white sand 1016, the relaxation flowers 9620, the flower culture 917 and the flower culture 33 are increased by 12.00%, 21.80%, 22.20%, 23.60%, 30.57% and 23.37% respectively compared with the control; side branch length, treatment increased by 12.30%, 22.71%, 11.25%, 14.31%, 16.00% and 19.38% respectively over control; total fresh mass, treatment increased 17.11%, 13.42%, 13.27%, 21.63%, 16.91% and 16.69% over control, respectively; total dry mass, treatment increased 34.59%, 47.90%, 12.30%, 34.79%, 26.95% and 11.24% over control, respectively.
Mycorrhizal dependencies of Lu Hua No. 8, medium flower No. 24, white sand 1016, yuhua 9620, huayu 917 and Huayu 33 are 25.70%, 32.29%, 10.95%, 25.20%, 20.89% and 10.08%, respectively, as shown in fig. 6.
Example 3
Effect of strain APS-1 spore suspension on peanut southern blight resistance
The pots were filled with moist field soil, inoculated with a spore suspension of about 900 strains of APS-1 at 2/3 of the height of the pot, after which the pots were filled with soil and an equal volume of water was inoculated as a control.
The peanut varieties of Luhua No. 8, zhonghua No. 24, baisha 1016, yuhua No. 9620, huayu 917 and Huayu 33 are sowed in flowerpots, 4 seeds are sowed in each flowerpot, and 3 peanut plants are reserved after emergence. 4 pots each were controlled and treated, repeated 3 times, all peanuts were placed in a plastic greenhouse and watered periodically. After 6 weeks, the basal part of the peanut stem is inoculated with oat grains full of southern blight hypha, and after 2 weeks, the occurrence of southern blight is investigated, and the control effect is calculated. Grading survey criteria for southern blight: stage 1: only the stems have lesions, grade 2: wilting and dying of the whole plant below 1/4, grade 3: whole plant 1/4-1/2 wilting and death, grade 4: and wilting and death of more than 1/2 of the whole plant. Disease index = Σ (value of each level×number of disease plants of each level)/(highest level×total number of investigation) ×100, incidence (%) = number of disease plants/total number of investigation×100%, control effect (%) = (control disease index-treatment disease index)/control disease index×100%.
The statistics show that strain APS-1 was able to significantly increase the resistance of 6 peanut varieties to southern blight compared to the control, as shown in fig. 7 and 8. Wherein, the indices of the conditions of the southern blight of luhua No. 8, medium flower No. 24, white sand 1016, yuhua 9620, hua yu 917 and hua yu 33 were reduced by 63.21%, 31.65%, 61.64%, 85.35%, 74.61% and 43.76% respectively, compared to the control (fig. 7, wherein, the difference is very significant at p <0.01 level); the incidence of southern blight was reduced by 63.04%, 30.12%, 58.82%, 78.19%, 54.89% and 53.34% respectively in the treatment compared to the control (fig. 7); the control effect is 89.14%, 48.81%, 65.50%, 55.23%, 58.24% and 43.76% respectively (figure 8).
The foregoing description is only a preferred embodiment of the present invention and is not intended to limit the present invention, but although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or that equivalents may be substituted for part of the technical features thereof. Any modification, equivalent replacement, variation, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Shandong province peanut institute
<120> A strain of Rhizopus delemar APS-1 and application thereof
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aaagttggac aaacttggtc atttagagga agtaaaagtc gtaacaaggt ttccgtaggt 120
gaacctgcgg aaggatcatt accgattttt agcggacctg atctttgatc atggtctcgc 180
gaaaacttgt atttaaaacc ccactcttat aaattgaatc attttatatt atgtatgtaa 240
taaataaaga tctctttcaa caacggatct cttgcctctc gcatcgatga acaacgtaac 300
gaagtgcgat aagtaatgtg aattgcataa ttccgtgaat catcgaatct ttgaacgcaa 360
attgcactct ctggtaaccc ggggagtatg cctgtttgag ggtcagtgtt aataaaaatc 420
ggtgcttggc aatttttgtg atgttccgga gtttgagata tcttatcttc tgggtttaag 480
aggcttaata ttgaccttct ttgtgcattt tagacgtaca taaatttttt ttattcgttt 540
atcttaatgc caaaatcaat tatatgcgac catatcgtgt ggtgccgtgc ctataaattt 600
ttcatgattt gacctcaaat caggtaggaa cacccgctga acttaagcat atcaataagc 660
ggaggaaaag aaaataacaa tgattcccct agtaactgcg agtgaagagg gacaagctca 720
aattttaaat ctatcgggtt ttacctgatc gagttgtaat ttgaagaagc gttttctgcg 780
tatttggtca gcctaagtcc tattgaatga ggcatcatgg agggtgataa tcccgtccgg 840
ttgatcattt gcgttttacg atacgctttc taagagtcaa gttgtttggg attgcagctt 900
aaaatgggag gtaaatttct cctaaggcta aataacggcg agagaccgat agcgaacaag 960
taccgtgagg gaaagatgaa aagaactttg aaaagagagt taaacagtac gtgaaattgt 1020
tgaaagggaa acgattgaag ccagtcgtac cttcgggtaa tcagcctttc gggtgcgatt 1080
ctgtggagtg tgaggagctt aaacaccttc atgttttgca tattcgtact cttgggtgta 1140
cttgcccgtg tggttggtta acatcaattt tggtggtcat aaaatggctg ggggaatgta 1200
gcttcgatct tgtattgaag tgttatagcc ttcggtagat gtgatgatcg agattgagga 1260
ttgcaacgga tacccttcgg ggctacctgt ctggtctctg atcgttgctc tggtgccgaa 1320
agcttgctta cggttatcaa agtcgatggt caataggtta gaacgggtta aattcgt 1377
Claims (6)
1. The special-shaped rhizopus altaicus (Rhizophagus irregularis) APS-1 is characterized by being preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.40116.
2. The use of the rhizopus deleteri APS-1 according to claim 1 for controlling peanut southern blight.
3. Use according to claim 2, characterized in that the application form is a spore suspension of strain APS-1.
4. Use of the rhizopus deleteri APS-1 as defined in claim 1 for promoting peanut growth.
5. The use according to claim 4, characterized in that the form of application is a spore suspension of strain APS-1.
6. A microbial inoculum for preventing and treating peanut southern blight, the active ingredient of which is the rhizopus delemar APS-1 as defined in claim 1.
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| CN114921346B true CN114921346B (en) | 2024-05-03 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450536A (en) * | 2014-11-13 | 2015-03-25 | 山东农业大学 | Separation of arbuscular mycorrhizal fungi as well as preparation and application of arbuscular mycorrhizal fungi fungicide |
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| CN104450536A (en) * | 2014-11-13 | 2015-03-25 | 山东农业大学 | Separation of arbuscular mycorrhizal fungi as well as preparation and application of arbuscular mycorrhizal fungi fungicide |
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