CN116622523B - Blue fungus 401 and application thereof - Google Patents
Blue fungus 401 and application thereof Download PDFInfo
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- CN116622523B CN116622523B CN202310650447.2A CN202310650447A CN116622523B CN 116622523 B CN116622523 B CN 116622523B CN 202310650447 A CN202310650447 A CN 202310650447A CN 116622523 B CN116622523 B CN 116622523B
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Classifications
-
- 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
- C12N1/145—Fungal isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/05—Fruit crops, e.g. strawberries, tomatoes or cucumbers
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- 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
Abstract
The invention provides a blue fungus 401 and application thereof, belonging to the technical field of biological control, wherein the blue fungus 401 is preserved in the Guangdong province microorganism strain collection center, and the preservation number is GDMCCNo:63188; the blue fungus 401 is obtained by separating and screening from the roots of the vetiver; the bacteriostasis rate of the composition to banana fusarium wilt bacteria reaches 71.00+/-1.77; the blue fungus 401 can induce the obvious swelling, increase of branches, roughness, deformity and crosslinking deformation of the TR4 hyphae; expanding the mycelium tip to form a bubble shape; hyphae expand in the middle; the number of spores is increased, the surface of the spores is rough, deformed and recessed, and hyphae are broken; the fertilizer also has a certain growth promoting potential and has the capabilities of dissolving inorganic phosphorus, fixing nitrogen and producing siderophores; and the blue fungus 401 does not cause pathogenic effect on banana leaves, and is not a potential pathogenic bacterium of bananas; the invention provides new fungus resources for preventing and treating banana vascular wilt in later period.
Description
Technical Field
The invention belongs to the technical field of biological control, and particularly relates to a cyanobacteria 401 and application thereof.
Background
Bananas are important tropical and subtropical fruits in the world. Nowadays, bananas have become the fourth largest food crop following rice, wheat, corn in developing countries. Banana insect pests are important constraint factors in banana production, and can cause loss of banana yield and quality, and even regional and global banana industry fluctuation. Wherein banana wilt is a destructive disease which occurs most seriously in banana producing area and is most difficult to control. Banana vascular wilt is a soil-borne vascular bundle disease caused by fusarium oxysporum (Fusarium oxysporum f.sp.cube, foc). The classification of banana fusarium wilt is mainly based on the hazard condition of pathogenic bacteria on different types of banana strains and even different genus species, wherein 4 physiological species are classified into subtropical race 4 (STR 4) and tropical race 4 (TR4). However, fusarium oxysporum No. 4 physiological race tropical (Fusarium oxysporum f.sp.cubic therapeutic race 4, foc TR4) has wide hosts and large harm, is a strong pathogenic bacteria of banana vascular wilt, and has the greatest danger and destructiveness on banana production. Based on incomplete statistics, foc TR4 has been found in the major banana producing areas of the world and gradually expands worldwide from the sick area to the non-sick area, which has become an important limiting factor in the development of the banana industry worldwide. Banana wilt is used as a soil-borne disease, and bacterial bud-sucking, plant residue and bacterial soil can be used as infectious sources of pathogenic bacteria. At present, the prevention measures mainly comprise chemical agent prevention and treatment, disease-free tissue culture seedling, disease-resistant variety, agricultural improvement prevention and treatment, biological prevention and treatment and the like. Foc TR4 is limited in its ability to survive without banana as a host, and thus its risk of morbidity can be reduced by rotation between different crops and agronomic practices such as improving field sanitation. In addition, inhibition of banana vascular wilt can be achieved using chemical control, such as the use of bactericides and soil fumigants. Chemical control is the most widely used mode at present, but the repeated use of a large amount of chemical control is easy to cause environmental pollution and other problems. Another approach is to breed resistant breeds, which have been developed in some research progress for Foc TR1 resistant breeds (Arinaitwe I K, teo C H, kayat F, et al evaluation of banana germplasm and genetic analysis of an F1population for resistance to Fusarium oxysporum f.sp.cube race 1[ J ]. Euphytica,2019,215 (10): 175.), but to date, foc TR4 resistant breeds have not been bred. Among these measures, although the disease-free tissue culture seedlings have a certain effect, the cost is relatively high, and the cultivated bananas are triploid, so that the breeding of disease-resistant varieties is difficult, and the popularization of the measures is more restricted. Chemical control and agricultural improvement measures are difficult to achieve ideal effects due to some specific factors of soil-borne diseases. Biological control is a green way for preventing and controlling banana wilt, namely, antagonism and growth-promoting biological control bacteria are explored, and the biological control bacteria have the characteristics of remarkable capability of promoting plant growth or preventing and controlling soil-borne diseases, environmental friendliness, safety and no toxicity, and are widely applied to agricultural production.
Endophytic fungi have been proved in biological control of soil-borne pathogens of different crops, and are also an important source of biocontrol fungi, and are widely applied in the aspects of controlling plant diseases and promoting growth. Trichoderma reesei strain CSR-T-3 was found by Damodaran et al to control fusarium oxysporum banana-induced wilt with a high inhibition rate of 85.19% (Damodaran T, rajan S, muthakumarM, et al biological management of banana Fusarium wilt caused by Fusarium oxysporum f.sp.cubic mechanical race 4using antagonistic fungal isolate CSR-T-3 (Trichoderma reesei) [ J ]. Frontiers in Microbiology,2020, 11:595.); the plant endophyte can biologically control plant diseases through antagonism, competition colonization, plant disease resistance induction and other actions, and some plant endophytes can promote plant growth through nitrogen fixation, enhance plant resistance and indirectly exert biological control action on plant diseases. The partial antagonistic bacteria can be planted in rhizosphere and root, so that not only can the pathogen be inhibited, but also the host plant can be induced to generate disease resistance; yu and Teng et al found an antagonistic bacterial strain of Fusarium oxysporum, pseudomonas BAF.1, which inhibited Fusarium oxysporum by the resulting siderophore with a maximum inhibition of 95.24% (Yu S M, teng C Y, liang J S, et al Characterization of siderophore produced by Pseudomonas syringae BAF.1and its inhibitory effects on spore germination and myceliummorphology ofFusarium oxysporum [ J ]. Journal ofmicrobiology (Seoul, korea), 2017,55 (11): 877-884.). At present, no endophytic fungi capable of antagonizing banana vascular wilt have been found.
Disclosure of Invention
In view of the above, the present invention aims to provide a blue fungus 401 and application thereof, wherein the blue fungus 401 is isolated and screened from the roots of the vetch by taking the pathogenic bacteria of banana wilt as target bacteria, and has antagonistic and growth promoting effects on banana wilt, and has growth promoting capability while inhibiting the pathogenic bacteria of banana wilt.
The invention provides a blue fungus (Talaromyces oumae-annae) 401, wherein the blue fungus 401 is deposited in the Guangdong province microorganism strain collection center, and the deposit number is GDMCC No:63188.
the invention provides application of the cyanobacteria 401 in promoting plant growth.
Preferably, the cyanobacteria 401 promotes plant growth by phosphate solubilizing, nitrogen fixation and/or siderophore production.
Preferably, the plant is banana.
The invention provides application of the cyanobacteria 401 in preventing and treating banana vascular wilt.
Preferably, the pathogenic bacteria of banana vascular wilt are fusarium oxysporum No. 4 physiological race.
Preferably, the bananas are root irrigated with the fermentation broth of the cyanobacteria 401.
Preferably, the spore concentration of the fermentation broth is 10 7 ～10 9 cfu/mL。
Preferably, the root irrigation treatment is performed for 1-4 times, and the interval time between two adjacent root irrigation treatments is 6-8 d.
Compared with the prior art, the invention has the following beneficial effects: the invention takes pathogenic bacteria of banana vascular wilt as target bacteria, fungus strain blue fungus 401 with antagonism and growth promotion effects on banana vascular wilt is obtained by separating and screening from the roots of the photo-leaf vetch, and the bacteriostasis rate on banana vascular wilt reaches 71.00% +/-1.77; after the blue fungus 401 and the TR4 are opposite to each other for 7 days through a flat plate, the TR4 hyphae are induced to be obviously swollen, branches are increased, the hyphae are rough, malformed and crosslinked and deformed; expanding the mycelium tip to form a bubble shape; hyphae expand in the middle; the number of spores is increased, the surface of the spores is rough, deformed, dented and hyphae are broken.
Potted experiments show that the infection of the blue fungus 401 to the TR4 has an inhibition effect; the cyanobacteria 401 also has a certain growth promoting potential and has the capabilities of inorganic phosphorus dissolution, nitrogen fixation and siderophore production; and the blue fungus 401 does not cause pathogenic effect on banana leaves, and is not a potential pathogenic bacterium of bananas; the invention provides new fungus resources for preventing and treating banana vascular wilt in later period.
Description of biological preservation
Cyanobacteria (Talaromyces oumae-annae) 401 was deposited with the cantonese collection of microorganism strains under the accession number GDMCC No:63188; the preservation date is 2023, 2 and 22 days, and the preservation address is Guangdong university of Guangzhou City, mitsui No. 100, no. 59, guangdong university of 5.
Drawings
FIG. 1 is a schematic illustration of the inoculation location and sampling of antagonistic fungi for a dual culture assay;
FIG. 2 is a graph showing the bacteriostatic effect of different antagonistic strains on banana vascular wilt;
FIG. 3 shows the bacteriostatic effect of strain 401 on banana vascular wilt; wherein A is the diameter of TR4 under the double culture of 401 strain and TR4; b is the inhibition rate of 401 strain to TR4; the data for the different lowercase letters indicate a significant difference at the 0.05 level;
FIG. 4 shows the antagonistic effect of strain 401 on Fusarium oxysporum;
fig. 5 shows the antagonism effect of the strain 401 on fusarium oxysporum, wherein e and f are scanning electron microscope structures of banana fusarium wilt pathogenic bacteria TR4 hypha in the inhibition state of the strain 401, and g and h are scanning electron microscope structures for normal growth of control banana fusarium wilt pathogenic bacteria TR4 hypha;
FIG. 6 is a graph showing the effects of phosphate solubilizing, nitrogen fixation and siderophore production by strain 401;
FIG. 7 shows the morphology and microstructure of strain 401;
FIG. 8 is a genetic developmental tree (A) and fungal ITS-PCR electrophoretogram (B) based on the r DNA-ITS gene sequence of strain 401;
FIG. 9 shows the biocontrol effect on banana leaf blades and the growth-promoting effect on banana by inoculating antagonistic 401 strain, wherein A is TR4; b is CK; c is TR4+401; d is 401;
FIG. 10 is the effect of inoculating an antagonistic bacterium on banana bulbs after inoculation, wherein A is TR4; b is CK; c is TR4+401; d is 401.
Detailed Description
The invention provides a blue fungus (Talaromyces oumae-annae) 401, wherein the blue fungus 401 is deposited in the Guangdong province microorganism strain collection center, and the deposit number is GDMCC No:63188; hereinafter, strain 401 is abbreviated.
In the invention, the strain 401 is separated from a sample of the root of the vetch, and is preserved after separation and purification. Strain 401 was grown in PDA medium, the colony morphology of the strain was observed, and microscopic morphology observation was performed using a scanning electron microscope strain. After the strain 401 is cultured for 14 days, the colony diameter is 76mm, and the colony morphology is observed, and hypha of the colony is found to be granular or flocculent, and the surface is flat; the mycelium appeared yellow, the back was dark red or white, and the colony edge was white in color. The microstructure finds that the mycelium part of the strain 401 is branched, bent and slender, the length is 31-45 mu m, the base part is 1.2-2 mu m, the mycelium is uniform, when the mycelium grows to a certain extent, a plurality of circular bulges are formed on the surface of the mycelium, and the size is about 0.5-1.5 mu m.
Extracting genome DNA of the strain 401 as a template, performing PCR amplification on the genome DNA of the strain 401 by using universal primers ITS 1and ITS4, sequencing, performing BLAST analysis on the obtained sequence in NCBI (www.ncbi.nlm.nih.gov) database, performing sequence homology comparison on other fungi, and determining the species classification status of the strain 401. The highest homology of strain 401 to Talaromyces coprophilus was found, up to 99.62%. The sequence with higher download similarity is constructed in GenBank by using MEGA7.0 software and a phylogenetic tree is constructed by using a maximum likelihood method, so that the strain 401 is identified at a molecular level, the strain 401 and the blue fungus are determined to be gathered into one, the support rate is 96 percent, and meanwhile, the antagonistic bacteria 401 is identified as the blue fungus (Talaromyces oumae-annae) by combining colony morphology and microscopic characteristic observation.
The invention also provides application of the cyanobacteria 401 in promoting plant growth.
In the present invention,the cyanobacteria 401 preferably promotes plant growth by phosphate solubilizing, nitrogen fixation and siderophore production; in the implementation process of the invention, the fermentation liquid of the cyanobacteria 401 is preferably used for root irrigation treatment of plants; the spore concentration of the fermentation broth is preferably 10 7 ～10 9 cfu/mL, more preferably 1 to 9X 10 8 cfu/mL; the fermentation broth is preferably obtained by culturing the cyanobacteria 401 in PDB broth. In the present invention, the plant is preferably banana. In the invention, the diameter value of the phosphate solubilizing ring of the strain 401 is 5.23+/-0.09 cm, and the diameter value of the nitrogen fixation ring is 4.03+/-0.12 cm; as a result of determining whether to produce siderophores by using a CAS dye solution staining culture plate, the yellow halo diameter of the strain 401 was found to be 6.13.+ -. 0.09cm; the strain 401 has the capabilities of fixing nitrogen, decomposing organic phosphorus and producing siderophores, and has great application potential in the aspect of growth promotion.
The invention also provides application of the cyanobacteria 401 in preventing and treating banana vascular wilt.
In the invention, the pathogenic bacteria of banana vascular wilt are preferably fusarium oxysporum No. 4 physiological race. In the implementation process of the invention, the fermentation liquor of the cyanobacteria 401 is preferably used for root irrigation treatment of bananas; the spore concentration of the fermentation broth is preferably 10 7 ～10 9 cfu/mL, more preferably 1 to 9X 10 8 cfu/mL; the fermentation broth is preferably obtained by culturing the cyanobacteria 401 in PDB broth. In the present invention, the number of root irrigation treatments is preferably 1 to 4, more preferably 2 to 3; the interval time between two adjacent root irrigation treatments is preferably 6-8 d, and more preferably 7d. In the present invention, the cyanobacteria 401 preferably promotes banana plant growth by alleviating the inhibitory effect of the physiological race 4 of Fusarium oxysporum on banana plants.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Test materials
The root sample of the test light leaf vetch was sampled at 11 months of 2021, and the five-point sampling method was adopted to carry out (102 DEG 41'E and 25 DEG 28' N) on a green manure long-term positioning experiment base in Songming county of Kunming, yunnan province. The annual average air temperature and annual average precipitation in the area are respectively 11-22 ℃ and 899.8mm, and the banana is not planted and banana vascular wilt symptoms are not caused. The root sample of the vetch with the rhizosphere soil is put into a clean fresh-keeping bag which is prepared in advance, and the fresh-keeping bag is brought back to a laboratory for preservation at 4 ℃ for standby.
The banana vascular wilt is Fusarium oxysporum No. 4 physiological race Tropical (Fusarium oxysporum f.sp.cubic therapeutic race 4, foc TR4) and is separated, identified and stored from Brazil banana variety in Xishuangbanna planting field by the research team of the agricultural environment and resource institute of the agricultural academy of Yunnan.
Scanning electron microscopy (zeiss Sigma 300, berlin, germany); the test medium was potato agar medium (PDA, containing 200.0g potato per 1L, 20.0g glucose, 20.0g agar, no agar added as PDB medium), bengalhon medium (3.0 g sucrose per 1L, 3.0g sodium nitrate, 0.3g potassium dihydrogen phosphate, 0.7g dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 0.5g potassium chloride, 10.0g sodium chloride, 20.0g agar powder, 50.0mg chloramphenicol), picovskava inorganic phosphorus solid medium (Majorana, mo Weidi, zhao Dailin, etc.), isolation identification of phosphate-solubilizing antagonistic fungi and evaluation of their biocontrol against soft rot in kiwi [ J ]. Southern agricultural newspaper 2019,50 (8): 1748-1755 ], ashby nitrogen-free medium and CAS bilayer medium (Chen Yang. Isolation identification of nitrogen-fixing bacteria in rice and inhibition of strain BV 6. Beijing agriculture university [ D ]. 9).
Test method
Isolation, purification and preservation of antagonistic fungal strains
Collecting roots of the sweet potatoes, cleaning the surfaces of the roots with sterile water, airing the roots indoors, weighing l 0g of the cleaned plant roots under the sterile condition, sterilizing the surfaces with 75% ethanol, cutting the roots into small pieces of 2-3 cm by using a sterile blade, soaking the small pieces in 75% ethanol for 3min, and flushing the small pieces with sterile water for 2 times. Soaking in 0.1% mercuric chloride for 1min, washing with sterile water for 3 times, placing into a sterilized mortar filled with 9mL sterile water, adding slightly sterilized quartz sand, grinding, standing for 15min to obtain1mL of diluted to 10 -3 、10 -4 、10 -5 、10 -6 Concentration gradients, 0.1mL from each concentration gradient suspension was applied to PDA and Bengal medium plates with sterile applicator bars and incubated upside down at 28℃with sterile water from the last 1 wash of the sample as control.
The observation was continued for 1 to 2 days, and individual colonies were counted and continued to be observed. When colonies grow on the surface of the solid culture medium, respectively picking the colonies with different forms and sizes, inoculating the colonies to a new solid culture medium for culture, separating out strains with single forms, and purifying for 2 times when the colonies grow on the new solid culture medium, namely separating out a plurality of single strains. The single strain after purification is inoculated on PDA solid slant culture medium by inoculating needle to pick colony, and stored temporarily in refrigerator at 4 deg.C.
Antagonistic fungus strain primary screening
The primary screening was carried out by a plate counter method (Fan H, li S, zeng L, et al biological control of Fusarium oxysporum f.sp.fundamental therapeutic 4using natively isolated Bacillus spp.YN0904 and YN1419[J. Journal of Fungi,2021,7 (10): 795.), preparing PDA medium, pouring the plate for use, inoculating banana vascular wilt cake of 2mm diameter in the center of PDA medium with a sterile punch, and gently pressing to prevent dropping. Inoculating and screening fungus strains at a position 2.5cm apart from the fungus cake, placing the fungus strains on the same flat plate, sealing with a sealing strip, repeating for 3 times by taking the flat plate without inoculating antagonistic fungus strains to be screened as a control, placing the flat plate in an incubator, culturing at 28 ℃ for 72 hours, and primarily screening fungus strains with antibacterial effect.
Antagonistic fungus strain rescreening
For screening antagonistic Fungi with antifungal activity against Foc TR4, reference is made to the plate counter method of modified Li (Li S, he P, fan H, et al A Real-Time Fluorescent Reverse Transcription Quantitative PCR Assay for Rapid Detection of Genetic Markers' Expression Associated with Fusarium Wilt of Banana Biocontrol Activities in Bacillus [ J ]. Journal of Fungi,2021,7 (5): 353). Inoculating pathogenic bacteria cake of banana vascular wilt in center of newly prepared PDA culture medium plate cross line, inoculating fungus mycelium with antibacterial effect to 2.5cm of TR4 bacteria cake (shown in figure 1), culturing at 28deg.C for 7 days, and repeating for 3 times. And measuring the growth distance of pathogenic bacteria, and calculating the average antibacterial effect.
Inhibition ratio (%) = [ (control group pathogenic bacteria growth diameter-treatment group pathogenic bacteria growth diameter)/(control group pathogenic bacteria growth diameter-inoculation cake diameter) ] ×100%.
Determination of phosphorus-decomposing ability of endophyte
Preparing an inorganic phosphate-dissolving solid medium of Picovskava, taking antagonistic fungus cakes with the diameter of 2mm by using a sterile puncher, inoculating the antagonistic fungus cakes to the center of the inorganic phosphate-dissolving solid medium, and culturing for 7d in a culture box at the temperature of 28 ℃. The appearance of transparent rings around the colony shows that the strain has the capability of decomposing inorganic phosphorus (Mao Ting, mo Weidi, zhao Dailin, etc.), the separation and identification of phosphorus-decomposing antagonistic fungi and the biological control evaluation of kiwi soft rot [ J ]. Southern agricultural journal, 2019,50 (8): 1748-1755.).
Determination of nitrogen fixation capacity of endophyte
The separated endophytic fungi are inoculated on a solid Ashby nitrogen-free medium, antagonistic fungi cakes with the diameter of 2mm are inoculated at the center of the Ashby nitrogen-free medium by a sterile puncher, and the endophytic fungi are cultured for 7d in a 28 ℃ incubator. The appearance of transparent rings around the colony shows that the strain has the capability of fixing nitrogen (Chen Yang. Separation and identification of rice endophytic azotobacter and research on fungus inhibition mechanism of strain BV6 [ D ]. Nanjing: nanjing agricultural university, 2019.).
Detection of secretion ability of endophyte siderophore
Siderophores refer to a pair of Fe secreted by bacteria to the surrounding under iron-deficient conditions 3+ Substances with very strong specific chelation. The CAS plate method uses Chromium Azure S (CAS), ferric ion and cetyl trimethyl ammonium bromide (HTDMA) to form a sky blue complex, and the culture plate is stained with CAS dye solution, and when the strain releases ferric ion into the culture plate, the ternary complex is destroyed and turns yellow, i.e., yellow halos appear around the colonies. Recording the presence or absence of orange halos on the culture medium, and simultaneously measuring the diameter of single colony orange siderophore halos (Chen Yang. Isolation and identification of rice endophytic azotobacter and research on fungus inhibition mechanism of strain BV6 [ D)]NanjingUniversity of agriculture, 2019.).
Molecular biological identification of antagonistic fungal strains
The DNA of the antagonistic strain was extracted using TSINGKE plant DNA extraction kit (universal type). The universal primer ITS1/ITS4 amplifies the ITS sequence of the strain. The PCR reaction system (50. Mu.L) was: 1×TSE101 gold plate mix 45. Mu.L, ITS1 (10P) 2. Mu.L, ITS4 (10P) 2. Mu.L, and DNA template 1. Mu.L. Thermal cycling conditions for PCR: pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 56 ℃ for 10s, extension at 72 ℃ for 10s,35 cycles, warm bath at 72 ℃ for 5min and preservation at 4 ℃ for standby. The amplified PCR product was subjected to agarose gel electrophoresis (2. Mu.L of sample+6. Mu.L of bromophenol blue) at 300V for 12min to obtain an identification gel. The ready PCR product was sent to Beijing qingke biosciences, inc. for sequencing (sequencing primers ITS1/ITS 4). And then carrying out phylogenetic analysis, carrying out BLAST sequence homology comparison on ITS gene sequences of fungi on NCBI to obtain homologous sequences, and constructing a phylogenetic tree by adopting a MEGA7.0 adjacent method.
Antagonizing fungi to control banana fusarium wilt potted plant and measuring growth promoting effect of banana plants
Pretreatment of potted plants
Greenhouse potting experiments were performed from 8 months to 11 months of 2022. And transferring the Brazilian banana tissue culture seedlings into a sand matrix for domestication for 30 days. And 5-6 banana plants with leaves are selected for potting experiments, and the biological control and growth promotion effects of TR4 are evaluated.
Preparation of antagonistic strain fermentation stock solution
Inoculating the endophytic fungus colony into PDB liquid culture medium by using aseptic inoculating needle, shake culturing at 28deg.C and 180r/min for 72 hr to obtain fermentation broth, and regulating spore concentration to 1×10 by using blood cell counting plate 8 cfu/mL。
Preparation of TR4 fusarium oxysporum spore liquid fermentation liquor
Inoculating activated TR4 pathogenic bacteria mycelium into PDB liquid culture medium, shake culturing at 28deg.C and 180r/min for 72 hr, and filtering mycelium with four layers of sterile gauze to obtain TR4 spore suspension. Diluting with sterile water to 1×10 6 cfu/mL of TR4 spore solution, stored at 4deg.C for use.
Potted plant test design
And 5-6 banana plants with leaves are selected for potting experiments, and root injury treatment is carried out before root irrigation of each pot of banana seedlings. Positive control group 1 (TR 4): root irrigation treatment (50 mL) is carried out by using fusarium oxysporum spore fermentation liquid suspension with the concentration of 1 multiplied by 106 cfu/mL; negative control group 2 (CK): PDB liquid medium (50 mL); treatment group 1 (tr4+ antagonistic fungus): using fermentation solution (50 mL) of antagonistic strain and fusarium oxysporum spore fermentation liquor with concentration of 1 multiplied by 106 cfu/mL; treatment group 2 (antagonistic fungi): antagonistic strain fermentation solution (50 mL). After 7d, treatment groups 1and 2 were again root-irrigated with an equal amount of antagonistic fungal fermentation stock (50 mL). All treatments were repeated 3 times.
Evaluation of TR4 biological control effect and banana growth promoting effect
After 45d inoculation, reference is made to the method of Fan (Fan H, li S, zeng L, et al biological control of Fusarium oxysporum f.sp. fundamental biological race 4using natively isolated Bacillus spp.YN0904 and YN1419[J ]. Journal of fungi,2021,7 (10): 795.); the extent of onset of banana plant leaves and bulbs was investigated on a 5-grade scale of 0-4. Classifying leaf diseases: level 0: no symptoms; stage 1: the yellow withered area of the true leaves and the cotyledons is not more than 50% of the total area; 2 stages: the yellow withered areas of the true leaves and cotyledons exceeded 50% of the total area; 3 stages: leaf withering or death, only growth points survive; 4 stages: the whole plant seriously withers or dies. Grading bulb diseases: level 0: the bulb has no lesions; stage 1: the area of the bulb lesions is 1-10%; 2 stages: the area of the bulb lesions is 11-30%; 3 stages: the area of the bulb lesions is 31-50%; 4 stages: the areas of the bulb lesions are respectively more than 50 percent. The plant height, stem thickness, leaf number, leaf length, leaf width, fresh weight of the overground part and fresh weight of the underground part of each banana after each treatment are measured and recorded.
Morbidity index (%) = Σ (number of patients at each stage×number of relative stages)/(total number of investigation×number of highest disease stage) ×100
Control effect (%) = (control disease index-treatment disease index)/control disease index×100
Data analysis
Data processing and analysis were performed using Excel 2010 and SPSS 22.0 software. Analysis was performed using 0 rib 2021 software.
Results and analysis
Screening of antagonistic fungal strains
Different fungus strains separated and purified on roots of the vetch are subjected to a counter culture test with pathogenic bacteria of banana vascular wilt by adopting a flat plate counter method, and preliminary screening is carried out to obtain 3 fungus strains with obvious antagonism effect, namely 401, 403 and 406 strains respectively. The obtained 3 antagonistic fungi are subjected to a bacteriostasis rate rescreening test, and the 401 fungi strain has the highest bacteriostasis effect, the bacteriostasis rate reaches 71.00+/-1.77 (see fig. 2 and 3), and the inhibition effect is stable and is obviously superior to 403 and 406 strains, so that the 401 strain is selected as a study object.
TR4 hyphae and conidia were observed by scanning electron microscopy. The antibacterial effect of the 401 strain on banana wilt is shown in fig. 4, fig. 5, the TR4 is controlled to normally grow on banana wilt pathogenic bacteria hypha, the aerial hypha is obviously white flocculent, the hypha is milky white, smooth and uniform, the spore morphology is complete, and the number is normal (g and h in fig. 5). After 7 days of plate facing of strain 401 and TR4, strain 401 induced significant swelling of the TR4 hyphae, increased branching, coarseness, deformity, and cross-linking deformation of the hyphae (e, f, arrow 1 in FIG. 5). The mycelium tips dilated, in a bubble shape (e, f, arrow 2 in fig. 5), and the mycelium dilated in the middle (e, f, arrow 3 in fig. 5). The number of spores increased, the spore surface roughened, deformed, dented (e, f, arrow 4 in fig. 5), and the hyphae broken (e, arrow 5 in fig. 5).
Antagonistic fungus phosphate solubilizing, nitrogen fixation and iron carrier production capability determination
The phosphate solubilizing, nitrogen fixing and iron carrier producing capacities (shown as A and B in figure 6) of the strain 401 are detected by adopting a flat plate phosphate solubilizing ring method, a flat plate nitrogen fixing ring method and a CAS dye liquor staining flat plate method, wherein the diameter value of the phosphate solubilizing ring of the strain 401 is 5.23+/-0.09 cm, and the diameter of the nitrogen fixing ring is 4.03+/-0.12 cm; the culture plate was stained with CAS dye to determine whether strain 401 produced siderophores, and when the strain released ferric ions into the culture plate, the ternary complex was destroyed and changed to yellow, i.e., yellow halos appeared around the colonies, resulting in the finding that strain 401 had a yellow halo diameter of 6.13.+ -. 0.09cm. Comprehensive analysis shows that the strain 401 has the functions of fixing nitrogen, decomposing organic phosphorus and producing iron carrier; besides good effect of antagonizing pathogenic bacteria of banana vascular wilt, the compound has great potential in growth promotion.
Fungus identification
Morphological identification
The isolated strain 401 was grown on PDA medium at 28 c, the colony morphology of the strain was observed (fig. 7), and the strain 401 was observed microscopically by scanning electron microscopy (fig. 7). After the strain 401 is cultured for 14 days, the colony diameter is 76mm, and the colony morphology is observed by naked eyes, so that hyphae of the strain are granular or flocculent, and the surface is flat; the mycelium appeared yellow, the back was dark red or white, and the colony edge was white in color. The microstructure discovers that the mycelium of the strain 401 is partially branched, bent and slender, has the length of 31-45 mu m, the base part is 1.2-2 mu m wide, the mycelium is uniform, and when the mycelium grows to a certain extent, a plurality of circular bulges are formed on the surface of the mycelium, and the size of the bulges is about 0.5-1.5 mu m.
Molecular biological identification
Extracting genomic DNA of 401 antagonistic fungus strain as template, using general primer ITS 1and ITS4 to make PCR amplification, using 1% agarose gel to make electrophoresis detection, making photographing in imaging system, observing to obtain a clear and bright electrophoresis band, and using Marker for electrophoresis as 5000bp, and making gene fragment size obtained by 401 amplification be 500-750 bp.
Sequencing was performed by Beijing, the resulting sequences were BLAST analyzed in NCBI (www.ncbi.nlm.nih.gov) database and compared for sequence homology to other fungi to determine the species classification status of each strain. The homology of 401 and Talaromyces coprophilus is found to be highest, and the homology is up to 99.62%; the sequence with higher download similarity is constructed by using MEGA7.0 software in GenBank and a phylogenetic tree is shown as A in figure 8 by using a maximum likelihood method, thus the 401 antagonistic fungus strain is identified on a molecular layer, the 401 antagonistic fungus and the blue fungus are gathered into one, the support rate is 96 percent, and meanwhile, the 401 antagonistic fungus is identified as the blue fungus (Talaromyces oumae-annae) by combining colony morphology and microscopic characteristic observation.
Antagonizing fungus to prevent banana fusarium wilt potted plant
After 45d inoculation, the leaves of banana (TR 4, A in FIG. 9) inoculated with TR4 broth alone turned yellow, the plants developed poorly, the lower part She Kusi dropped off, and the incidence was higher; while most of the leaves in treatment group 1 (tr4+401, G in fig. 9) remained healthy, the plants grew well, no obvious symptoms of disease, and the plant growth status was not significantly different from that of banana seedlings in control group (CK, B in fig. 9), indicating that antagonism of 401 against fungi clearly plays a role in inhibiting TR4 infection. While treatment group 2 (antagonistic fungus 401 fermentation solution, H in fig. 9) was not ill, indicating that antagonistic fungus 401 did not produce a pathogenic effect on banana leaves, not a potential pathogenic bacteria of banana.
Furthermore, further observations were made of banana bulbs, in which the banana bulbs inoculated with TR4 fermentation broth alone (TR 4, a in fig. 10) showed obvious symptoms in comparison with the control plants (CK, B in fig. 10), the bulbs inoculated with TR4 appeared to appear as brown-black infected areas, the bulbs were of obvious browning in longitudinal section, basal decay, the bulbs in treatment group 1 (tr4+401) had different disease conditions (G in fig. 10), and the tr4+401 treatment group banana bulbs were of longitudinal section without disease. Whereas banana bulbs of treatment group 2 (antagonistic fungus 401 fermentation solution, H in FIG. 10) did not have an onset, the bulb longitudinal profile was similar to that of the blank control, and no onset area was observed.
In conclusion, the antagonistic fungus 401 can obviously inhibit infection of TR4 on bananas, reduce the disease condition of banana wilt, and treat the bananas independently without causing pathogenic effect on the bananas.
Proved by potted plant test investigation, the antagonistic fungus 401 has remarkable inhibition effect on TR4 pathogenic bacteria (table 1), the prevention and treatment effect of the corm is 96.876%, and the disease index is remarkably lower than that of the individual TR4 treatment.
TABLE 1 biological control of TR4 in potting experiments by antagonistic fungus 401
Note that: the different lower case letters after the same column of data represent significant differences (P < 0.05).
Antagonizing the growth promoting effect of fungi on banana
The results of banana seedling growth promotion test on antagonistic fungus 401 (table 2, fig. 9 and fig. 10) show that compared with a blank control, the independent inoculation of antagonistic fungus 401 fermentation liquor has no obvious effect on banana plant growth, and the leaf number of the banana plant is reduced by 401 treatment; compared with the independent inoculation of the TR4 fermentation liquor treatment, the inoculation of 401 and the TR4 fermentation liquor treatment can obviously promote the growth of banana seedlings, respectively improve the banana plant height (44.2%), the stem circumference (22.7%), the leaf number (67.7%), the leaf length (33.3%), and the fresh weight of the overground part (84.5%) and the underground part (317.3%), which shows that the antagonistic fungus 401 can obviously reduce the inhibition effect of TR4 on the banana growth so as to promote the banana growth, and the treatment groups inoculated with the antagonistic fungus and the TR4 fermentation liquor have insignificant difference compared with the independent inoculation of the antagonistic fungus treatment, can more show that the antagonistic fungus reduces the inhibition effect of TR4 on the banana growth, and can be used as an efficient disease-resistant and growth-promoting strain resource.
TABLE 2 antagonism of the effects of fungus 401 on banana growth promotion
Note that: the different lower case letters after the same column of data represent significant differences (P < 0.05).
As can be seen from the above examples, the bacteriostasis rate of the blue-like bacteria 401 provided by the invention to banana fusarium wilt bacteria reaches 71.00+/-1.77; the blue fungus 401 can induce the obvious swelling, increase of branches, roughness, deformity and crosslinking deformation of the TR4 hyphae; expanding the mycelium tip to form a bubble shape; hyphae expand in the middle; the number of spores is increased, the surface of the spores is rough, deformed and recessed, and hyphae are broken; the blue fungus 401 plays a remarkable role in inhibiting the infection of TR4; the blue-like bacteria 401 also has remarkable growth promoting potential and has the capability of decomposing inorganic phosphorus, fixing nitrogen and producing siderophores; and the blue fungus 401 does not cause pathogenic effect on banana leaves, is not a potential pathogenic bacterium of bananas, and has wide application prospect.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (6)
1. A cyanobacterium (talaromyceoumae-annae) 401, wherein said cyanobacterium 401 is deposited with the cantonese microbiological bacterial collection center under the accession number GDMCCNo:63188.
2. use of the cyanobacteria 401 of claim 1 for promoting banana growth, wherein the cyanobacteria 401 promotes banana growth by reducing the inhibition of banana growth by the physiological race tropical species of fusarium oxysporum 4.
3. The use according to claim 2, wherein the cyanobacteria 401 promote plant growth by phosphate solubilizing, nitrogen fixation and/or siderophore production.
4. The use of the cyanobacterium 401 according to claim 1 for preventing and treating banana vascular wilt, wherein the pathogenic bacteria of banana vascular wilt is fusarium oxysporum No. 4 physiological race tropical.
5. The use according to claim 4, characterized in that the bananas are root-irrigated with the fermentation broth of the cyanobacteria 401; the spore concentration of the fermentation liquid is 10 7 ～10 9 cfu/mL。
6. The use according to claim 5, wherein the number of root irrigation treatments is 1-4 and the interval between two adjacent root irrigation treatments is 6-8 d.
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