CN112941141A - Fungus for inhibiting growth of rice blast fungus and blocking melanin secretion of rice blast fungus - Google Patents
Fungus for inhibiting growth of rice blast fungus and blocking melanin secretion of rice blast fungus Download PDFInfo
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- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 title claims abstract description 120
- 241000233866 Fungi Species 0.000 title claims abstract description 62
- 230000028327 secretion Effects 0.000 title claims abstract description 57
- 230000012010 growth Effects 0.000 title claims abstract description 52
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 29
- 230000000903 blocking effect Effects 0.000 title claims abstract description 13
- 241001330975 Magnaporthe oryzae Species 0.000 title claims description 20
- 241000209094 Oryza Species 0.000 claims abstract description 64
- 235000007164 Oryza sativa Nutrition 0.000 claims abstract description 64
- 235000009566 rice Nutrition 0.000 claims abstract description 64
- 241000894006 Bacteria Species 0.000 claims abstract description 59
- 230000000443 biocontrol Effects 0.000 claims abstract description 50
- 230000002503 metabolic effect Effects 0.000 claims abstract description 26
- 239000007788 liquid Substances 0.000 claims abstract description 24
- 230000000694 effects Effects 0.000 claims abstract description 20
- 239000001963 growth medium Substances 0.000 claims abstract description 15
- 230000005764 inhibitory process Effects 0.000 claims abstract description 14
- 238000009630 liquid culture Methods 0.000 claims abstract description 14
- 239000007787 solid Substances 0.000 claims abstract description 14
- 238000002474 experimental method Methods 0.000 claims abstract description 6
- 238000007789 sealing Methods 0.000 claims description 12
- 241001344131 Magnaporthe grisea Species 0.000 claims description 9
- 230000008859 change Effects 0.000 claims description 9
- 238000011282 treatment Methods 0.000 claims description 9
- 235000021186 dishes Nutrition 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 230000000844 anti-bacterial effect Effects 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 239000003550 marker Substances 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 3
- 239000002207 metabolite Substances 0.000 claims description 2
- 239000012528 membrane Substances 0.000 claims 1
- 230000009036 growth inhibition Effects 0.000 abstract description 3
- 244000005700 microbiome Species 0.000 abstract description 2
- 239000012530 fluid Substances 0.000 abstract 2
- 230000001580 bacterial effect Effects 0.000 description 9
- 241000196324 Embryophyta Species 0.000 description 6
- 208000031888 Mycoses Diseases 0.000 description 5
- 244000053095 fungal pathogen Species 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 230000003042 antagnostic effect Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 244000000004 fungal plant pathogen Species 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 230000003993 interaction Effects 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 241000223602 Alternaria alternata Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 206010061217 Infestation Diseases 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 208000035415 Reinfection Diseases 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000008099 melanin synthesis Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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Abstract
The invention provides a fungus for inhibiting the growth of rice blast fungi and blocking melanin secretion of the rice blast fungi, and relates to the technical field of microorganisms. The analysis of the biocontrol bacterium effect on the growth inhibition of the rice blast fungi comprises the following specific experimental methods: analyzing the growth amount of biocontrol bacteria (SS 7) on rice blast bacteria and the melanin secretion inhibition effect by plate confronting culture; step two, analyzing the growth amount and melanin secretion inhibiting effect of the biocontrol strain (SS 7) metabolic liquid on rice blast fungi by PDA solid culture; and step three, analyzing the growth and melanin secretion inhibiting effect of the biocontrol strain (SS 7) metabolic liquid on the rice blast fungi by PDA liquid culture. The biological bacteria (SS 7) can obviously inhibit and even block the melanin secretion of the rice blast bacteria, the biocontrol bacteria has obvious growth inhibition effect on the rice blast bacteria and obvious inhibition effect on the melanin secretion in a PDA flat plate bacteriostasis test, and when the addition amount of the biocontrol bacteria metabolic fluid in a PDA culture medium reaches 75 percent, the biocontrol bacteria metabolic fluid not only can inhibit the growth of the rice blast bacteria, but also can completely block the melanin secretion of the rice blast bacteria.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a fungus for inhibiting the growth of rice blast fungi and blocking the melanin secretion of the rice blast fungi.
Background
Fungal diseases of plants are one of three diseases (bacterial diseases, fungal diseases and viral diseases) in plant cultivation, and all cultivation areas in the world occur, so that the occurrence of the fungal diseases can cause yield reduction to different degrees, and even the fungal diseases are completely harvested in severe cases. The main conditions of the cycle and serious occurrence of plant fungal diseases are primary infection and secondary infection, and the primary infection is based on the safe overwintering quantity of pathogenic bacteria; the re-infection is based on the condition of suitable and rapid propagation after the initial infection. In plant pathogenic fungi, many fungi secrete melanin in the growth process, such as Pyricularia oryzae and Alternaria alternata of tomato early blight. Whether initially infected or re-infected with these melanin-secreting pathogens, melanin production is a prerequisite for the pathogenic fungi to complete the plant infestation. Therefore, the inhibition of melanin secretion is a direct means for blocking pathogenic fungi from infecting plants, and is the simplest and most effective method for preventing plant diseases. So far, no report about biocontrol bacteria for inhibiting the secretion of melanin of plant pathogenic fungi exists.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a fungus for inhibiting the growth of rice blast fungi and blocking the melanin secretion of the rice blast fungi, so that the goal of inhibiting the melanin secretion of most pathogenic fungi is expanded, and the problem of biocontrol bacteria which do not inhibit the melanin secretion of plant pathogenic fungi is solved.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: a fungus for inhibiting the growth of rice blast fungus and blocking the melanin secretion of the rice blast fungus comprises the following specific experimental methods:
analyzing the growth amount of biocontrol bacteria (SS 7) on rice blast bacteria and the melanin secretion inhibition effect by plate confronting culture;
step two, analyzing the growth amount and melanin secretion inhibiting effect of the biocontrol strain (SS 7) metabolic liquid on rice blast fungi by PDA solid culture;
and step three, analyzing the inhibition effect of the biocontrol bacteria (SS 7) on the growth of the rice blast fungi and the melanin secretion by PDA liquid culture.
Preferably, the first step of analyzing the growth amount of rice blast fungi and the melanin secretion inhibiting effect of biocontrol bacteria (SS 7) by plate confrontation culture is embodied as follows:
select the culture dish that the specification is unanimous, divide the three-section equally with marker pen equidistance with culture dish bottom diameter, at superclean bench, interior aseptic hole puncher is respectively in living control fungus (SS 7) and the magnaporthe grisea fungus of activated edge of colony of fungus and get the hypha piece of equidimension, receive living control fungus (SS 7) and magnaporthe grisea fungus piece simultaneously the mark point department on the diameter of flat board, make 2 fungus pieces 3cm apart from each other, the sealing film seals and inverts 25 ℃ constant temperature cultivation in the constant temperature incubator, observe, every group repeats 3 dishes, treat that two bacterial colony hypha contact back, observe two bacterial colony morphologies and two interactions between the bacterial colony hypha, calculate the size that two growth volume averages are the bacterial colony diameter, calculate the bacteriostasis rate: p ═ D1-D2)/D1 × 100%;
in the formula: d1 represents control pure growth (cm); d2 represents the treatment pure growth (cm);
and simultaneously observing the change of melanin secretion of the rice blast fungi, and analyzing the difference by referring to a control.
Preferably, the second step of analyzing the growth amount and the melanin secretion inhibiting effect of the biocontrol bacteria (SS 7) metabolic liquid on the magnaporthe grisea PDA solid culture specifically comprises the following steps:
transferring the activated biocontrol bacteria (SS 7) into a liquid PDA culture medium, carrying out constant-temperature oscillation culture until the pH value is constant (about 20D), filtering the biocontrol bacteria (SS 7) metabolic liquid through four layers of gauze, respectively adding the biocontrol bacteria (SS 7) metabolic liquid into a conventional PDA solid culture medium according to the concentration gradients of 0%, 25%, 50%, 75% and 100%, taking equal-size mycelium blocks at the edge of the same colony of the activated rice blast strain in an ultraclean workbench by using a puncher, respectively inoculating the mycelium blocks into solid culture dishes with different treatments, marking after sealing films are sealed, repeating the concentration gradients for 3 times, measuring the colony diameter of the rice blast bacteria every 3D after the rice blast bacteria obviously germinate (7D), and calculating the bacteriostasis rate by P ═ D1-D2)/D1 multiplied by 100%;
and simultaneously observing the change of melanin secretion of the rice blast fungi, and analyzing the difference by referring to a control.
Preferably, the third step of analyzing the growth and melanin secretion inhibiting effect of the biocontrol bacteria (SS 7) on rice blast fungi by PDA liquid culture is as follows:
adding biocontrol bacteria (SS 7) metabolic liquid into a conventional PDA liquid culture medium according to the concentration gradients of 0%, 25%, 50%, 75% and 100%, taking mycelium blocks with the same size at the edge of the same colony of an activated rice blast strain in a super clean workbench by using a puncher, respectively inoculating the mycelium blocks into liquid culture media with different treatments, inoculating 4 mycelium blocks into each bottle, sealing a sealing film to seal a mark, repeating the concentration gradients for 3 times, observing every 3d after the rice blast bacteria obviously germinate (7d), collecting mycelium in the culture solution by using a filter paper filtration method when culturing for 20d, placing in a drying box at 80 ℃ for drying, analyzing the mass of the mycelium weighed by a balance, and calculating the growth amount;
and simultaneously observing the change of melanin secretion of the rice blast fungi, and analyzing the difference by referring to a control.
(III) advantageous effects
The invention provides a fungus for inhibiting the growth of rice blast fungi and blocking the melanin secretion of the rice blast fungi. The method has the following beneficial effects:
the biological bacteria (SS 7) can obviously inhibit and even block the secretion of melanin of pathogenic fungi, the biocontrol bacteria has obvious growth inhibition effect on rice blast fungi and obvious inhibition effect on the secretion of the melanin in a PDA flat plate bacteriostasis test, and when the addition amount of the biocontrol bacteria metabolic solution in a PDA culture medium reaches 75 percent, the biocontrol bacteria metabolic solution not only can inhibit the growth of the rice blast fungi, but also can completely block the secretion of the melanin.
Drawings
FIG. 1 is a graph showing the results of experiments on the effect of biocontrol bacteria (SS 7) on the growth of Pyricularia oryzae and melanin secretion during plate-confrontation culture according to the present invention;
FIG. 2 is a graph showing the experimental results of the effect of the metabolite of biocontrol bacteria (SS 7) on the growth of Pyricularia oryzae and the secretion of melanin during the solid culture of PDA in accordance with the present invention;
FIG. 3 is a graph showing the results of experiments on the effect of biocontrol bacteria (SS 7) on the growth of Pyricularia oryzae and melanin secretion in the liquid culture of PDA according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example (b):
as shown in the figure, the embodiment of the invention provides a fungus for inhibiting the growth of rice blast fungi and blocking the melanin secretion of the rice blast fungi, which comprises the following specific experimental methods:
analyzing the growth amount of biocontrol bacteria (SS 7) on rice blast bacteria and the melanin secretion inhibition effect by plate confronting culture;
step two, analyzing the growth amount and melanin secretion inhibiting effect of the biocontrol strain (SS 7) metabolic liquid on rice blast fungi by PDA solid culture;
and step three, analyzing the growth and melanin secretion inhibiting effect of the biocontrol strain (SS 7) metabolic liquid on the rice blast fungi by PDA liquid culture.
The first step is to analyze the growth amount of the biocontrol strain (SS 7) on the rice blast bacteria and the melanin secretion inhibiting effect through plate confronting culture, which is embodied as follows:
select the culture dish that the specification is unanimous, divide the three-section equally with marker pen equidistance with culture dish bottom diameter, at superclean bench, interior aseptic hole puncher is respectively in living control fungus (SS 7) and the magnaporthe grisea fungus of activated edge of colony of fungus and get the hypha piece of equidimension, receive living control fungus (SS 7) and magnaporthe grisea fungus piece simultaneously the mark point department on the diameter of flat board, make 2 fungus pieces 3cm apart from each other, the sealing film seals and inverts 25 ℃ constant temperature cultivation in the constant temperature incubator, observe, every group repeats 3 dishes, treat that two bacterial colony hypha contact back, observe two bacterial colony morphologies and two interactions between the bacterial colony hypha, calculate the size that two growth volume averages are the bacterial colony diameter, calculate the bacteriostasis rate: p ═ D1-D2)/D1 × 100%;
in the formula: d1 represents control pure growth (cm); d2 represents the treatment pure growth (cm);
and simultaneously observing the change of melanin secretion of the rice blast fungi, and analyzing the difference by referring to a control.
After the antagonistic line is generated, the hyphae on the side of the antagonistic line in the culture dish can cover the surface of the rice blast fungus to continue growing along the antagonistic line with the lapse of the culture time, and the hyphae of the biocontrol bacterium (SS 7) has obvious inhibition effect on the growth of the rice blast fungus and the melanin secretion.
And step two, the growth amount and the melanin secretion inhibiting effect of the biocontrol strain (SS 7) metabolic liquid on the magnaporthe grisea PDA solid culture are specifically embodied as follows:
transferring the activated biocontrol bacteria (SS 7) into a liquid PDA culture medium, carrying out constant-temperature oscillation culture until the pH value is constant (about 20D), filtering the biocontrol bacteria (SS 7) metabolic liquid through four layers of gauze, respectively adding the biocontrol bacteria (SS 7) metabolic liquid into a conventional PDA solid culture medium according to the concentration gradients of 0%, 25%, 50%, 75% and 100%, taking equal-size mycelium blocks at the edge of the same colony of the activated rice blast strain in an ultraclean workbench by using a puncher, respectively inoculating the mycelium blocks into solid culture dishes with different treatments, marking after sealing films are sealed, repeating the concentration gradients for 3 times, measuring the colony diameter of the rice blast bacteria every 3D after the rice blast bacteria obviously germinate (7D), and calculating the bacteriostasis rate by P ═ D1-D2)/D1 multiplied by 100%;
and simultaneously observing the change of melanin secretion of the rice blast fungi, and analyzing the difference by referring to a control.
Note: different capital letters indicate heteropolar significance at P <0.01 levels, lower case letters indicate heteropolar significance at P <0.05 levels
Compared with a control, the growth of the rice blast bacteria in the culture medium of the metabolic solution with different concentrations is obviously different along with the extension of the culture time, the larger the concentration is, the smaller the colony diameter of the rice blast bacteria is, the obvious bacteriostatic effect is achieved, the difference is extremely obvious, and the bacterial colonies of the rice blast bacteria in the culture dish with the concentrations of 50% and 75% have the phenomenon that hypha melanin is obviously degraded in a concentric circle manner; the inhibition effect of the metabolic liquid on the rice blast fungi is continuously constant in the later period of the test, and the inhibition effect is very obvious different all the time.
Step three, analyzing the growth and melanin secretion inhibiting effect of the biocontrol strain (SS 7) on the rice blast fungus through PDA liquid culture, and concretely embodying the following steps:
adding biocontrol bacteria (SS 7) metabolic liquid into a conventional PDA liquid culture medium according to the concentration gradients of 0%, 25%, 50%, 75% and 100%, taking mycelium blocks with the same size at the edge of the same colony of an activated rice blast strain in a super clean workbench by using a puncher, respectively inoculating the mycelium blocks into liquid culture media with different treatments, inoculating 4 mycelium blocks into each bottle, sealing a sealing film to seal a mark, repeating the concentration gradients for 3 times, observing at intervals of 3d after the rice blast bacteria obviously germinate (7d), collecting the mycelium in the culture solution by using a filter paper filtration method when culturing for 20d, placing the mycelium in a drying box at 80 ℃ for drying, measuring the mass of the mycelium by using an electronic analytical balance, and comparing data; and simultaneously observing the change of melanin secretion of the rice blast fungi, and analyzing the difference by referring to a control.
And (3) measuring the dry weight of the hyphae after the liquid culture of the rice blast fungi for 20 days, wherein the biocontrol strain (SS 7) metabolic liquid with different proportions has obvious inhibition effect on the growth of the rice blast fungi hyphae, the inhibition effect is concentration-related, the higher the concentration is, the more obvious the antibacterial effect is, and the antibacterial difference is extremely obvious. The melanin secretion of Magnaporthe grisea is completely blocked when the addition amount of biocontrol bacteria (SS 7) metabolic liquid reaches 75%.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (4)
1. The fungus for inhibiting the growth of rice blast fungi and blocking the melanin secretion of the rice blast fungi is characterized by comprising the following specific experimental methods:
analyzing the growth amount of biocontrol bacteria (SS 7) on rice blast bacteria and the melanin secretion inhibition effect by plate confronting culture;
step two, analyzing the growth amount and melanin secretion inhibiting effect of the biocontrol strain (SS 7) metabolic liquid on rice blast fungi through PDA solid culture;
and step three, analyzing the growth and melanin secretion inhibiting effect of the biocontrol strain (SS 7) metabolic liquid on the rice blast fungi by PDA liquid culture.
2. The fungus for inhibiting the growth of Pyricularia oryzae and blocking the melanin secretion according to claim 1, wherein the first step of analyzing the growth amount of Pyricularia oryzae and the melanin secretion-inhibiting effect of biocontrol bacteria (SS 7) by plate confrontation culture comprises:
the culture dish of 9cm internal diameter is got, divide the three-section equally with marker pen with culture dish bottom diameter, take equidimension hypha piece with aseptic hole puncher respectively at activated biocontrol fungus (SS7 number) and the magnaporthe grisea fungus colony edge in superclean bench, connect biocontrol fungus (SS7 number) and magnaporthe grisea fungus piece simultaneously to dull and stereotyped mark point department on the diameter, make 2 fungus pieces 3cm apart from each other, the sealing membrane seals and inverts 25 ℃ constant temperature cultivation in the constant temperature incubator, the repeated 3 dishes of every group, treat that two colony hypha contact back, observe the interact between two colony forms and two colony, calculate the size that two growth volume averages are the colony hypha diameter, calculate the antibacterial rate: p ═ D1-D2)/D1 × 100%;
in the formula: d1 represents control pure growth (cm); d2 represents the treatment pure growth (cm);
and simultaneously observing the change of melanin secretion of the rice blast fungi, and analyzing the difference by referring to a control.
3. The fungus for inhibiting the growth of Pyricularia oryzae and blocking the melanin secretion according to claim 1, wherein the second step of analyzing the yield of Pyricularia oryzae (SS 7) and the effect of the metabolite of biocontrol fungus on the growth of Pyricularia oryzae and the melanin secretion inhibition by PDA solid culture is embodied as follows:
transferring the activated biocontrol bacteria (SS 7) into a liquid PDA culture medium, carrying out constant-temperature oscillation culture until the pH value is constant (about 20D), filtering the biocontrol bacteria (SS 7) metabolic liquid through four layers of gauze, respectively adding the biocontrol bacteria (SS 7) metabolic liquid into a conventional PDA solid culture medium according to the concentration gradients of 0%, 25%, 50%, 75% and 100%, taking equal-size mycelium blocks at the edge of the same colony of the activated rice blast strain in an ultraclean workbench by using a puncher, respectively inoculating the mycelium blocks into solid culture dishes with different treatments, marking after sealing films are sealed, repeating the concentration gradients for 3 times, measuring the colony diameter of the rice blast bacteria every 3D after the rice blast bacteria obviously germinate (7D), and calculating the bacteriostasis rate by P ═ D1-D2)/D1 multiplied by 100%;
and simultaneously observing the change of melanin secretion of the rice blast fungi, and analyzing the difference by referring to a control.
4. The fungus for inhibiting the growth of Pyricularia oryzae and blocking the melanin secretion according to claim 1, wherein the inhibitory effect of biocontrol bacteria (SS 7) on the growth of Pyricularia oryzae and the melanin secretion is determined by the following steps:
adding biocontrol bacteria (SS 7) metabolic liquid into a conventional PDA liquid culture medium according to the concentration gradients of 0%, 25%, 50%, 75% and 100%, taking mycelium blocks with the same size at the edge of the same colony of an activated rice blast strain in a super clean workbench by using a puncher, respectively inoculating the mycelium blocks into liquid culture media with different treatments, inoculating 4 mycelium blocks into each bottle, sealing a sealing film to seal a mark, repeating the concentration gradients for 3 times, observing at intervals of 3d after the rice blast bacteria obviously germinate (7d), collecting the mycelium in the culture solution by using a filter paper filtration method when culturing for 20d, placing the mycelium in a drying box at 80 ℃ for drying, measuring the mass of the mycelium by using an electronic analytical balance, and comparing data;
and simultaneously observing the change of melanin secretion of the rice blast fungi, and analyzing the difference by referring to a control.
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CA2760153A1 (en) * | 2009-04-27 | 2010-11-11 | Alex Chenchik | Reagents and methods for producing bioactive secreted peptides |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CA2760153A1 (en) * | 2009-04-27 | 2010-11-11 | Alex Chenchik | Reagents and methods for producing bioactive secreted peptides |
CN110249379A (en) * | 2017-01-24 | 2019-09-17 | 隆萨有限公司 | The method and system of industrial maintenance is carried out using the display of virtual or augmented reality |
US20210251237A1 (en) * | 2018-07-25 | 2021-08-19 | Regents Of The University Of Minnesota | Platform for developing soil-borne plant pathogen inhibiting microbial consortia |
Non-Patent Citations (2)
Title |
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SAIMA KHALID等: "Chemical signals driving bacterial–fungal interactions", 《ENVIRONMENTAL MICROBIOLOGY》 * |
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