CN116606748B - Penicillium simplicissimum 204 and application thereof - Google Patents
Penicillium simplicissimum 204 and application thereof Download PDFInfo
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- CN116606748B CN116606748B CN202310646639.6A CN202310646639A CN116606748B CN 116606748 B CN116606748 B CN 116606748B CN 202310646639 A CN202310646639 A CN 202310646639A CN 116606748 B CN116606748 B CN 116606748B
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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
- A01N63/36—Penicillium
-
- 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
- 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
-
- 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
- C12R2001/80—Penicillium
-
- 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 penicillium 204 and application thereof, belonging to the technical field of biological control, wherein the penicillium 204 is preserved in the microorganism strain collection of Guangdong province, and the preservation number is GDMCCNo:63187; the penicillium simplicissimum 204 is obtained by separating and screening from the roots of the photic sweet potatoes; the bacteriostasis rate of the bacterial strain on banana fusarium wilt reaches 69.83+/-0.75; the penicillium 204 can cause the obvious swelling of the TR4 hyphae, the increase of branches, uneven thickness of the hyphae and expansion of the hyphae in the middle; spore number is increased, aggregation and hypha rupture; potted experiments show that Jian Qing mould 204 has an inhibition effect on the infection of TR4; the fertilizer also has a certain growth promoting potential and has the capabilities of dissolving inorganic phosphorus, fixing nitrogen and producing siderophores; moreover, the penicillium simplicissimum 204 does not cause pathogenic effect on banana leaves, and is not a potential pathogenic bacteria 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 penicillium simplicissimum 204 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 penicillium simplicissimum 204 and application thereof, wherein pathogenic bacteria of banana vascular wilt are used as target bacteria, fungus strain penicillium simplicissimum 204 with antagonistic and growth promoting effects on banana vascular wilt is obtained by separating and screening from roots of vetiver, and the fungal strain penicillium simplicissimum 204 has growth promoting capabilities while inhibiting the pathogenic bacteria of banana vascular wilt.
The invention provides a penicillium simplicissimum (Penicillium simplicissimum) 204, wherein Jian Qing mold 204 is deposited in the Guangdong province microorganism strain collection center, and the deposit number is GDMCC No:63187.
the invention provides application of the penicillium simplicissimum 204 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 Jian Qing mould 204.
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.
The invention provides application of the penicillium simplicissimum 204 in promoting plant growth.
Preferably, the Jian Qing mold 204 promotes plant growth by phosphate solubilizing, nitrogen fixation and siderophore production.
Preferably, the plant is banana.
Preferably, the Jian Qing mold 204 promotes banana plant growth by reducing the inhibition of banana plants by fusarium oxysporum No. 4 physiological race.
Compared with the prior art, the invention has the following beneficial effects: the invention takes pathogenic bacteria of banana fusarium wilt as target bacteria, fungus strain penicillium simplicissimum 204 with antagonism and growth promotion effects on banana fusarium wilt is obtained by separating and screening from the roots of the photo-leaf vetch, and the bacteriostasis rate on banana fusarium wilt reaches 69.83% +/-0.75; after the penicillium simplicissimum 204 and the TR4 are opposite to each other for 7 days through the flat plate, the hyphae of the TR4 can be caused to be obviously swollen, the branches are increased, the thickness of the hyphae is uneven, and the hyphae are expanded in the middle; spore number is increased, aggregation and hypha rupture; potted experiments show that Jian Qing mould 204 has an inhibition effect on the infection of TR4; the penicillium simplicissimum 204 also has a certain growth promoting potential and has the capabilities of inorganic phosphorus dissolution, nitrogen fixation and siderophore production; moreover, the penicillium simplicissimum 204 does not cause pathogenic effect on banana leaves, and is not a potential pathogenic bacteria of bananas; the invention provides new fungus resources for preventing and treating banana vascular wilt in later period.
Description of biological preservation
Penicillium sp (Penicillium simplicissimum) 204 was deposited with the Cantonese microorganism strain collection under the accession number GDMCC No:63187; 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 204 on banana vascular wilt; wherein A is the diameter of TR4 under double culture of 204 strain and TR4; b is the inhibition rate of 204 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 204 on Fusarium oxysporum;
fig. 5 shows the antagonism effect of the strain 204 on fusarium oxysporum, wherein c and d are scanning electron microscope structures of banana fusarium wilt pathogenic bacteria TR4 hypha in the inhibition state of the strain 204, and g and h are scanning electron microscope structures for normal growth of the banana fusarium wilt pathogenic bacteria TR4 hypha;
FIG. 6 is a graph showing the effects of phosphate solubilizing, nitrogen fixation and siderophore production of strain 204;
FIG. 7 shows the morphology and microstructure of strain 204;
FIG. 8 is a genetic developmental tree (A) and fungal ITS-PCR electrophoretogram (B) based on the r DNA-ITS gene sequence of strain 204;
FIG. 9 shows the biocontrol effect on banana leaves and the growth-promoting effect on banana by inoculating antagonistic 204 strain, wherein A is TR4; b is CK; c is TR4+204; d is 204;
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+204; d is 204.
Detailed Description
The invention provides a penicillium (Penicillium simplicissimum) 204, wherein the Jian Qing mold 204 is deposited in the Guangdong province microorganism strain collection center, and the deposition number is GDMCC No:63187; hereinafter, strain 204 is abbreviated.
In the invention, the strain 204 is separated from a sample of the root of the vetch, and is preserved after separation and purification. Strain 204 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. The bacterial colony has a diameter of 66mm after the bacterial strain 204 is cultured for 14d, the bacterial colony is micro-villus, the surface of the bacterial colony is loose and has dry powder and is accompanied by a small amount of raised mycelium, concentric ring lines or a small amount of radial wrinkles exist, the edge of the bacterial colony can show ring-like white halos, the bacterial colony is light black and green, and the back surface of the bacterial colony is milky white; the microstructure is found that the mycelium of the No. 204 strain is branched and slender partially, the end part is narrowed, the length is 35-40 mu m, the base part is 2.1-3 mu m wide, the thickness is uniform, the mycelium surface is smooth, and no spore structure is found.
Extracting genome DNA of the strain 204 as a template, performing PCR amplification on the genome DNA of the strain 204 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 204. The highest homology of strain 204 to Penicillium ochrochloron was found, up to 99.82%. 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 204 is identified at a molecular level, the strain 204 and penicillium simplicissimum are determined to be gathered into one, the support rate is 99 percent, and meanwhile, the antagonistic bacteria No. 204 are identified as penicillium simplicissimum (Penicillium simplicissimum) by combining colony morphology and microscopic characteristic observation.
The invention provides application of the penicillium simplicissimum 204 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 liquid of the Jian Qing mould 204 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 Jian Qing mould 204 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.
The invention also provides application of the penicillium simplicissimum 204 in promoting plant growth.
In the present invention, the Jian Qing mold 204 preferably promotes plant growth by phosphate solubilizing, nitrogen fixation and siderophores; the plant is preferably banana. In the invention, the diameter of the nitrogen fixation ring of the strain 204 is 2.13+/-0.41 cm, and the diameter value of the phosphorus dissolving ring is 4.73+/-0.15 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 204 was found to be 5.27.+ -. 0.15cm; the strain 204 has the capabilities of fixing nitrogen, decomposing organic phosphorus and producing iron carrier, has good effect of antagonizing banana vascular wilt pathogenic bacteria, and has great potential in growth promotion.
In the present invention, the P.succinogenes 204 preferably promotes banana plant growth by reducing the inhibitory effect of F.oxysporum No. 4 physiological race on banana plants. In the present invention, the specific operation of the application in promoting the growth of banana plants is consistent with the operation of the application in controlling banana vascular wilt as described above, and will not be described herein.
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. Reuse ofSoaking in 0.1% mercuric chloride for 1min, washing with sterile water for 3 times, placing into a sterilized mortar filled with 9mL of sterile water, adding a little sterilized quartz sand, grinding uniformly, standing for 15min, taking 1mL, and diluting 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 was made to modified Li (Li S, he P, fan H, et al A Real-Time Fluorescent Reverse Transcription Quantitative PCR Assay for Rapid Detection ofGenetic Markers) , Expression Associated with Fusarium Wilt of Banana Biocontrol Activities in Bacillus[J].Journal of Fungi,2021,7(5):353. ) The plates of the plate are opposite to each other. 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. Record orange on the mediumThe presence or absence of halos, and the diameter of single colony orange siderophore halos were measured simultaneously (Chen Yang. Isolation and identification of Azotobacter chroodii in rice and research on fungal inhibition mechanism of BV6 strain [ D)]Nanjing, nanjing university 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 sieving with four layers of sterile gauzeThe mycelium was filtered to give a 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): with a concentration of 1X 10 6 Performing root irrigation treatment (50 mL) on cfu/mL of fusarium oxysporum spore fermentation liquor suspension; negative control group 2 (CK): PDB liquid medium (50 mL); treatment group 1 (tr4+ antagonistic fungus): with a concentration of 1X 10 6 cfu/mL Fusarium oxysporum spore broth suspension (50 mL) +antagonistic strain fermentation solution (50 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 204, 205 and 206 strains. And (3) performing a bacteriostasis rate rescreening test on the obtained 3 antagonistic fungi. The 204 fungus strain has the highest antibacterial effect, the antibacterial rate reaches 69.83% +/-0.75 (see fig. 2 and 3), and the inhibition effect is stable and is obviously superior to that of 205 and 206 strains. Thus, 204 strain was selected as the subject.
TR4 hyphae and conidia were observed by scanning electron microscopy. The antibacterial effect of the 204 strain on banana wilt is shown in fig. 4, fig. 5, the TR4 control banana wilt pathogenic bacteria mycelium grows normally, the aerial hyphae are obviously white flocculent, the hyphae are milky white, smooth and uniform in color, the spore morphology is complete, and the number is normal (g and h in fig. 5). After 7 days of the plate facing the TR4 strain 204, the strain 204 resulted in a significant swelling of the TR4 hyphae, increased branching and uneven hyphae thickness (c, d, arrow 1 in FIG. 5). The hyphae dilate in the middle (c, d, arrow 3 in fig. 5). The number of spores increased and aggregated (d, arrow 4 in fig. 5). Hyphae break (c, 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 204 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 204 is 4.73+/-0.15 cm, and the diameter of the nitrogen fixing ring is 2.13+/-0.41 cm; the culture plate was stained with CAS dye to determine whether strain 204 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 204 had a yellow halo diameter of 5.27.+ -. 0.15cm. Comprehensive analysis shows that the strain 204 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 204 strain was grown on PDA medium at 28 ℃ to observe colony morphology of the strain (fig. 7), and microscopic morphology observation was performed on the 204 strain using a scanning electron microscope (fig. 7). 204 bacterial strains are cultured for 14 days, the colony diameter is 63mm, the colony morphology is observed with naked eyes, the colony is micro-villus, the surface is loose and has dry powder and is accompanied by a small amount of raised mycelium, concentric ring lines or a small amount of radial wrinkles exist, the edge can present ring-like white halos, the colony is light black and green, and the back surface belt is milky white; the microstructure is found that the mycelium of the No. 204 strain is branched and slender partially, the end part is narrowed, the length is 35-40 mu m, the base part is 2.1-3 mu m wide, the thickness is uniform, the mycelium surface is smooth, and no spore structure is found.
Molecular biological identification
Extracting 204 genome DNA of 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 size of gene fragment obtained by 204 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 204 and Penicillium ochrochloron is found to be highest and reaches 99.82%; 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, so that the 204 antagonistic fungus strain is identified on a molecular layer, the 204 antagonistic fungus and the penicillium simplicissimum are gathered together, the support rate is 99 percent, and meanwhile, the 204 antagonistic fungus is identified as the penicillium simplicissimum (Penicillium simplicissimum) 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+204, C 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 the 204 antagonism fungus clearly has a certain inhibition effect on TR4 infection. While treatment group 2 (antagonistic fungus 204 fermentation solution, D in fig. 9) was not ill, indicating that antagonistic fungus 204 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 significant symptoms in the bulbs inoculated with TR4, brown-black infected areas in the bulb color, apparent browning of the bulb longitudinal section, basal decay, different disease conditions in the bulbs in treatment group 1 (tr4+204, C in fig. 10), a part of the disease areas in the tr4+204 treatment group banana bulbs longitudinal section, and the area of the lesions of the bulbs was about 1 to 10%, but the disease areas were controlled to a certain extent and were not more than 10%, compared with the control plants (CK, B in fig. 10). Whereas banana bulbs of treatment group 2 (antagonistic fungus 204 fermentation solution, D in FIG. 10) were free of disease, the bulbs were similar in longitudinal profile compared to the blank, and no disease area was observed.
In conclusion, the antagonistic fungus 204 can inhibit the infection of the TR4 on the bananas to a certain extent, reduce the disease condition of banana vascular wilt, and treat the bananas independently without causing pathogenic effect on the bananas and without causing pathogenicity on the bananas.
Proved by potted plant test investigation, the antagonistic fungus 204 has remarkable inhibition effect on TR4 pathogenic bacteria (table 1), the prevention and treatment effect of the corm is 65.623%, 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 204
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 the banana seedling growth promotion test on the antagonistic fungus 204 (table 2, fig. 9 and fig. 10) show that the independent inoculation of the antagonistic fungus 204 fermentation broth has no significant effect on the banana plant growth compared with the blank control; compared with the single inoculation of the TR4 fermentation liquor treatment, the inoculation of 204 and the TR4 fermentation liquor treatment can obviously promote the growth of banana seedlings, respectively improve the banana plant height (41.6%), the stem circumference (16.7%), the leaf number (52.2%), the leaf length (24.8%), and the fresh weight of the overground part (82.7%) and the underground part (217.2%), which shows that the antagonistic fungus 204 can reduce the inhibition effect of TR4 on the banana growth within a certain range so as to promote the banana growth, and the difference between the treatment groups of the inoculation antagonistic fungus and the TR4 fermentation liquor is not obvious compared with the single inoculation antagonistic fungus treatment, can more show that the inhibition effect of the antagonistic fungus on the banana growth by reducing the TR4 can be used as an efficient disease-resistant and growth-promoting strain resource.
TABLE 2 antagonism of the effects of fungi 204 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 antibacterial rate of the Jian Qing mould 204 provided by the invention on banana fusarium wilt bacteria reaches 69.83% +/-0.75; the penicillium 204 can cause the obvious swelling of the TR4 hyphae, the increase of branches, uneven thickness of the hyphae and expansion of the hyphae in the middle; spore number is increased, aggregation and hypha rupture; potted experiments show that Jian Qing mould 204 has an inhibition effect on the infection of TR4; the fertilizer also has a certain growth promoting potential and has the capabilities of dissolving inorganic phosphorus, fixing nitrogen and producing siderophores; moreover, the penicillium simplicissimum 204 does not cause pathogenic effect on banana leaves, is not a potential pathogenic bacteria 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 (9)
1. Penicillium simplicissimum (L.) persPenicillium simplicissimum) 204, wherein the Jian Qing mold 204 is deposited with the cantonese collection of microorganism strains under the accession number GDMCC No:63187.
2. use of the Jian Qing mould 204 of claim 1 for controlling banana vascular wilt; the pathogenic bacteria of banana vascular wilt are physiological race tropical strains of fusarium oxysporum No. 4.
3. Use according to claim 2, characterized in that the bananas are root-irrigated with the fermentation broth of the Jian Qing mould 204.
4. The use according to claim 3, wherein the spore concentration of the fermentation broth is 10 7 ～10 9 cfu/mL。
5. The use according to claim 3 or 4, wherein the root irrigation is performed 1 to 4 times, and the interval between two adjacent root irrigation is 6 to 8 days.
6. Use of the Jian Qing mould 204 of claim 1 for promoting plant growth.
7. The use according to claim 6, wherein the Jian Qing mould 204 promotes plant growth by phosphate solubilizing, nitrogen fixation and siderophores.
8. The use according to claim 6 or 7, wherein the plant is banana.
9. The use according to claim 8, wherein the Jian Qing mould 204 promotes banana plant growth by reducing the inhibition of banana plants by fusarium oxysporum No. 4 physiological race tropical.
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| CN101735955A (en) * | 2009-03-13 | 2010-06-16 | 南京信息工程大学 | Method for producing simple penicillium and metabolins thereof and application thereof |
| CN104419646A (en) * | 2013-09-04 | 2015-03-18 | 中国科学院天津工业生物技术研究所 | Penicillium simplicissimum capable of biologically synthesizing 1,2-testololactone and synthesis method of 1,2-testololactone |
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| CN101735955A (en) * | 2009-03-13 | 2010-06-16 | 南京信息工程大学 | Method for producing simple penicillium and metabolins thereof and application thereof |
| CN104419646A (en) * | 2013-09-04 | 2015-03-18 | 中国科学院天津工业生物技术研究所 | Penicillium simplicissimum capable of biologically synthesizing 1,2-testololactone and synthesis method of 1,2-testololactone |
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| The Plant Growth-Promoting Fungus Penicillium simplicissimum GP17-2 Induces Resistance in Arabidopsis thaliana by Activation of Multiple Defense Signals;Md. Motaher Hossain等;《Plant Cell Physiol.》;第48卷(第12期);第1724-1736页 * |
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