CA2347706A1 - Utilization of an antifungal protein from streptomyces tendae against plant pathogenic fungi - Google Patents
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Abstract
The invention relates to the use of an antifungal protein (AFP) having a molecular weight of approximately 10kDa from Streptomyces tendae against plant pathogenic fungi from the Ascomycetes family.
Description
WO 00/229:32 PCT/EP99/07364 Description Utilization of an antifungal protein from Streptomyces tendae 'ag<~inst plant pathogenic fungi The present invention relates to the use of an antifunga=_ protein (AFP) having a molecular weight of approx. 10 kDa from Streptomyces tendae against plant pathogenic: fungi fr.~om the Ascomycetes family.
Antifungal. proteins have been described for some time, some of these proteins also being termed PR proteins (plant pathogenesis--related proteins) in the case of plants (Bol, J.F. & Linthorst, H.J.M. (1990), Annu.
Rev. Phytopathol., 28, 113). These proteins are formed by plants under strc=ss conditions, for example in the case of viral or fungal infection, inducing an active defense mechanism of the plant which is termed "induced resistance" (Lindhorst, H.J.M., (1991) Cri. Rev. Plant Sci., 10(2), 123). :3ome of these proteins have, for example, a chitin<~se activity or (3-1,3-glucanase activity.
Proteins with an antifungal activity have already been detected from microorganisms too, some of these proteins also showing chitinase activity or (3-1,3-glucanase activity. Other proteins, in contrast, act via an interaction ofthe fungal cell wall.
For example, an antifungal protein (AFP) having a size of approx. 10 kDa which is active against the fungal species Paecilomyce;s varriotii, Byssoclamis nivea, Pencillium puberulum and Eupenicillium terrum has been isolated from Streptomyces tendae. However, the action was restricted to these species. Other species such as, for example, Paec.ilomyces carneus, Paecilomyces lilacenus, PeniciLlium chrysogenum or Penicillium claviforme are not inhibited. In addition, the use of AFP is not justified commercially either since the fungal species mentioned are not plant pathogenic.
The object of the present invention was therefore to find an antifungal protein which is active against plant pathogenic fungi.
Surprisin~~ly, it has now been found that AFP from Streptomy~~es tendae is active against plant pathogenic fungi from the Ascomycetes family.
The subject-matter of present the invention is therefore the use of- an antifungal protein (AFP) with a molecular weight of approx. 10 kDa from Streptomyces tendae against plant pathogenic fungi from the AscomycetE~s family, in particular against Botrytis cinera.
The antif:ungal prot=ein mentioned contains a typical amino-terminal. signal sequence, i.e. a hydrophilic N-terminu;~ is followed by a hydrophobic transmembrane region, which cause extracellular secretion. Moreover, two form, are known, namely a shorter form with a molecular weight o:~ approx. 9 860 Da and a longer form with a moJ_ecular weight of approx. 10 300 Da.
Since AFP is secreted by Streptomyces tendae into the culture medium, it can be isolated for example directly from the' Streptomyces tendae culture filtrate, following methods k:gown to the skilled worker. As an alternati~re, AFP can be produced by genetic engineering, the genre isolated preferably being cloned into a so-called multicopy vector, for example p1J702 (Hopwood, D.A. et a,_. (1985) Genetic Manipulation of Streptomyc:es. A Laboratory Manual. Norwich, U.K.) and using this construc=tion to transform a suitable strain, for example thF~ non-nikk;~mycin-producing strain Streptomyc:es tendac: NP9. An example of an AFP-encoding nucleic acid is shown in SEQ ID No. 1. When this preferred transform<~nt was grown, for example, for 7 days in :?00 ml of nutrient solution, approx. 6 mg of AFP were obtained, whi~~h corresponds to 30 mg of AFP/1.
The production of AFP by genetic engineering via a preferably controlled fermentation, is therefore especially preferred.
For the use in accordance with the invention, AFP can be applied directly for example in the form of a formulation together with preferably at least one additiona7_ auxiliary. To this end, for example, plants attacked by fungi or endangered by fungal attack are sprayed with the formulation described. The AFP-concentration of the formula i.s generally from approx.
10 ~g/ml up to approx. 500 mg/ml. Suitable auxiliaries are, for example, protease inhibitors, stabilizers such as glycerol, sucro:~e, salts, solvents or generally known substances from the field of crop protection.
So-called transgenic plants which are capable of producing AFP themselves are another possibility of providing protection against fungal infection.
Another embodiment :is therefore the use according to the invention of AFP, the AFP being formed in a transgenic plant. 'rhe transgenic plant is, preferably, genetically engineered maize, cotton, potato, banana, Arabidopsis, casava, tobacco, oilseed rape (canola), potato, sugar beet, cereals such as, for example, wheat, barley or oats, strawberries, vegetables such as, for example, cabbage, legumes such as, for example, peas or beans, tomato, lettuce or melon.
To generate a transgenic plant, a nucleic acid encoding AFP, for example ir_ 'she form of naked DNA, viral DNA or RNA, or in the form of plasmid DNA, is introduced into a plant cell. An e:Kample of an AFP-encoding nucleic acid is ~;hown in SEQ ID No. 1. However, the present invention also covers those nucleic acids which differ from the nucleic acid sequence of SEQ ID No. 1 owing to the degeneracy of the genetic code, but which encodes the same AFP amino acid sequence. Moreover, the invention covers thaw mutants or variants of the nucleic acid of SE;Q ID No. 1 which encode an AFP
protein which is active against plant pathogenic fungi of the Ascomycetes family, in particular against Botrytis cinera. 'Ih.ese include, for example, fusion proteins of the AFP ;pratein with other foreign proteins or an AFP protein with an N-terminally deleted methionine. Other examples of variants are nucleic acids which hybridize an the stringent conditions with the nucleic acid of SEQ ID No. 1. The stringent hybridization conditior~s can be determined for example by Sambrook, J. et al. in Molecular Cloning, A
Laboratory Manual, 2nd Edition, Cold Spring Habour Laboratory Press, 1989. Transgenic plants are subsequently regenerated from the transformed plant cells. Preferably, an AFP-encoding nucleic acid can be introduced into the plant cell by means of recombinant agrobacteria, by e:lE>ctroporation, by bombardment with microparticles and/or by means of polyethylene glycol.
The infection with recombinant Agrobacterium tumefaciens bacteria in plant cells of dicots is described, for examp7_e~, by Klee, H. et al.. (1997) Annu.
Rev. Plant Physiol. 38, 467 or EP-B2-0122791). The infection with re:ombinant Agrobacterium tumefaciens bacteria in plant cells of monocots is described, for example, by Ishida, 'Y. et al. (1996) Nature Biotech.
14, 745 with reference to maize (Zea mays L.). The agrobacteria used fcr this purpose have a T-DNA into which the AFP-expressing gene and, if appropriate, a suitable promoter, for example a plant phaseolin promoter (see, for example, EP-B2-0122791) or a viral 35S promoter (see, fcr example, Ishida, Y. et al.
WO 00/229:32 - 5 - PCT/EP99/07364 (1996), above) had been inserted. The T-DNA can be transferred into plant cells for example by coculturing the recornbinant agrobacteria together with immature embryos of the plant in question (see, for example, Ishida, '~. et a1. (1996), above). To select the transformed plant cc=_lls, it is preferred to use T-DNA
construct; which are capable of expressing, in the plant cell, a resistance gene from the AFP gene to be expressed.. A suitable resistance gene is, for example, the gene encoding phosphinothricin acetyltransferase (see, for example, Ishida, Y. et al. (1996}, above).
Thus, successfully transformed plant cells can be selected for example by means of the corresponding antibiotic phosphinothricin. The regeneration of a plant from transformed plant cells is generally known (see, for example, ~~agi et al. (1995), Nature Biotech, 13, 481-485, Ishida, Y. et al. (1996), above, or Schopke et al. (1996;x, Nature Biotech, 14, 731-735.
In addition to the use of Agrobacterium tumefaciens as transformation means, other transformation methods are known and suitable, such as, for example, electro-poration, the bombardment with microparticles or the use of polyethylene glycol (see, for example, Potrykos, (1991) Annu. Rev. Plant Physiol. Mol. Biol., 42, 205;
Estruch, J.J. et al. (1997), Nature Biotech., 15, 137-141 or Dingermann, 'T. (1995) BIOforum, 18, 252). For example, casava (Schopke et al. (1996), above) or banana (Sagi, et a:l. (1995), above) have already been transformed by bombarding plant cells with DNA-coated particles. In this method too, the AFP gene to be expressed is cloned into a suitable vector which preferably has a resistance gene allowing the subsequent selection of the transformed plant cells.
The recombinant erector is preferably applied to tungsten particles with which, for example, suspensions of embryogenic cel~.s are bombarded (see, for example, Sagi, et al. (195), above). The cell suspensions treated thus are sL.bsequently selected for transformed plant ce7.ls using a suitable antibiotic, for example phosphinothricin, and a transgenic plant is regenerated therefrom, for example as already described above.
A considerable advantage of the present invention is that AFP is active against plant pathogenic fungi from the Ascomycetes family, in particular against Botrytis Cinera, which have .already developed a resistance in particular to one or more crop protection agents.
The examples which follow and the figure are intended to describe the invention in greater detail without imposing any limitation:
Sequence description SEQ ID No. 1 shows a nucleic acid sequence encoding an ar~tifungal (223) protein (AFP) from Streptomyces tendae.
Examples Example 1:
To prepare test plates, 50 ~1 of a spore suspension of Botrytis cinerae were added to 100 ml of test agar (20 g/1 malt extra~~t, 10 g/1. glucose, 2 g/1 yeast extract, 0.5 ammor~:ium sulfate, i5 g/1 agar, pH 6.0).
25 ml of this were poured into Petri dishes. The final spore concentratiorv was 105 spores/ml. Sterile anti-biotic test disks (Sc:~leicher and Schuell, Dassel, Frg) 0.5 cm in diameter were subsequently applied to a test plate. Then, 5, 10, 15 and 20 ail of the AFP solution (100 mg/ml lyophili.z~~d culture filtrate of S. tendae-transformants) were subsequently pipetted onto the test disks and the test plate was incubated for 4 days at 30°C. The diameter of the inhibitory zone was 0.7 cm for 5 ~1 of AFP solution, 1 cm diameter for 10 ~1, 1.3 cm for 15 ul ofsolution and 1.9 cm for 20 ~1.
Example 2:
To prepare test plates, 50 ~l of a spore suspension of Botrytis cinerae ZF 3629 (resistant to BCM (Benlat, BenomylTM, Hoechst Sc:~ering AgrEvo GmbH, Frankfurt, carbendazem) were added to 100 ml of test agar (20 g/1 malt extract, 10 g,Jl glucose, 2 g/1 yeast extract, 0.5 ammonium :sulfate, 15 g/1 agar, pH 6.0). 25 ml of this were pou:=ed into Petri dishes. The final spore concentration was :LOS spores/ml. Sterile antibiotic test disks. (Schleic:h~=r and Schuell, Dassel, Frg) 0.5 cm in diameter were swbsequently applied to a test plate.
Then, 5, 10, 15 and 20 ~1 of the AFP solution (100 mg/ml lyophilized culture filtrate of S. tendae-transformants) were subsequently pipetted onto the test disks and the test plate was incubated for 4 days at 30°C. The diameter of the inhibitory zone was 0.9 cm for 5 ~tl of AFP solution, 1.3 cm diameter for 10 ul, 1.9 cm for 15 ~1 of ~~olution and 2.2 cm for 20 ul.
Example 3 - Improved production by fermentation:
In a flask equipped with a hose, 100 ml of the preculture medium (103 g/1 sucrose, 20 g Tryptic Soy Broth (Oxoid, Wesel), 10 g/1 MgCl~ and 10 g/1 yeast extract) were inoculated with an agar section of strain S. tendae. The culture was grown for 5 days at 28°C at 200 rpm on an orbit:~l shaker.
10 1 of the production medium (103 g/1 sucrose, 20 g Tryptic Soy Broth (Uxoid, Wesel), 10 g/1 MgCl2, 10 g/1 yeast extract, 100 ~Cl desmophen, pH 7.5) were inoculated with 7_00 ml of the above-described preculture.
The fermenter employed was a Biostat E (Braun, Melsungenl . Fermentation was carried out for 4 days at 28°C, 700 rpm and 0.5 vvm of air. Thereupon, fermentation was stopped, and the culture supernatant was harvested by cE=_ntrifugation at 3000 rpm and 4°C.
The batch was subsequently dried by lyophilization. It was possible to employ the powder directly. If 15 ~l of the lyophilizate (1.00 mg/ml) were employed against Botrytis cinerae in an inhibitory-zone test, an inhibitory zone of 1.7 cm and, against Botrytis cinerae ZF 3629, an inhibitory zone of 2.3 cm were detected.
Antifungal. proteins have been described for some time, some of these proteins also being termed PR proteins (plant pathogenesis--related proteins) in the case of plants (Bol, J.F. & Linthorst, H.J.M. (1990), Annu.
Rev. Phytopathol., 28, 113). These proteins are formed by plants under strc=ss conditions, for example in the case of viral or fungal infection, inducing an active defense mechanism of the plant which is termed "induced resistance" (Lindhorst, H.J.M., (1991) Cri. Rev. Plant Sci., 10(2), 123). :3ome of these proteins have, for example, a chitin<~se activity or (3-1,3-glucanase activity.
Proteins with an antifungal activity have already been detected from microorganisms too, some of these proteins also showing chitinase activity or (3-1,3-glucanase activity. Other proteins, in contrast, act via an interaction ofthe fungal cell wall.
For example, an antifungal protein (AFP) having a size of approx. 10 kDa which is active against the fungal species Paecilomyce;s varriotii, Byssoclamis nivea, Pencillium puberulum and Eupenicillium terrum has been isolated from Streptomyces tendae. However, the action was restricted to these species. Other species such as, for example, Paec.ilomyces carneus, Paecilomyces lilacenus, PeniciLlium chrysogenum or Penicillium claviforme are not inhibited. In addition, the use of AFP is not justified commercially either since the fungal species mentioned are not plant pathogenic.
The object of the present invention was therefore to find an antifungal protein which is active against plant pathogenic fungi.
Surprisin~~ly, it has now been found that AFP from Streptomy~~es tendae is active against plant pathogenic fungi from the Ascomycetes family.
The subject-matter of present the invention is therefore the use of- an antifungal protein (AFP) with a molecular weight of approx. 10 kDa from Streptomyces tendae against plant pathogenic fungi from the AscomycetE~s family, in particular against Botrytis cinera.
The antif:ungal prot=ein mentioned contains a typical amino-terminal. signal sequence, i.e. a hydrophilic N-terminu;~ is followed by a hydrophobic transmembrane region, which cause extracellular secretion. Moreover, two form, are known, namely a shorter form with a molecular weight o:~ approx. 9 860 Da and a longer form with a moJ_ecular weight of approx. 10 300 Da.
Since AFP is secreted by Streptomyces tendae into the culture medium, it can be isolated for example directly from the' Streptomyces tendae culture filtrate, following methods k:gown to the skilled worker. As an alternati~re, AFP can be produced by genetic engineering, the genre isolated preferably being cloned into a so-called multicopy vector, for example p1J702 (Hopwood, D.A. et a,_. (1985) Genetic Manipulation of Streptomyc:es. A Laboratory Manual. Norwich, U.K.) and using this construc=tion to transform a suitable strain, for example thF~ non-nikk;~mycin-producing strain Streptomyc:es tendac: NP9. An example of an AFP-encoding nucleic acid is shown in SEQ ID No. 1. When this preferred transform<~nt was grown, for example, for 7 days in :?00 ml of nutrient solution, approx. 6 mg of AFP were obtained, whi~~h corresponds to 30 mg of AFP/1.
The production of AFP by genetic engineering via a preferably controlled fermentation, is therefore especially preferred.
For the use in accordance with the invention, AFP can be applied directly for example in the form of a formulation together with preferably at least one additiona7_ auxiliary. To this end, for example, plants attacked by fungi or endangered by fungal attack are sprayed with the formulation described. The AFP-concentration of the formula i.s generally from approx.
10 ~g/ml up to approx. 500 mg/ml. Suitable auxiliaries are, for example, protease inhibitors, stabilizers such as glycerol, sucro:~e, salts, solvents or generally known substances from the field of crop protection.
So-called transgenic plants which are capable of producing AFP themselves are another possibility of providing protection against fungal infection.
Another embodiment :is therefore the use according to the invention of AFP, the AFP being formed in a transgenic plant. 'rhe transgenic plant is, preferably, genetically engineered maize, cotton, potato, banana, Arabidopsis, casava, tobacco, oilseed rape (canola), potato, sugar beet, cereals such as, for example, wheat, barley or oats, strawberries, vegetables such as, for example, cabbage, legumes such as, for example, peas or beans, tomato, lettuce or melon.
To generate a transgenic plant, a nucleic acid encoding AFP, for example ir_ 'she form of naked DNA, viral DNA or RNA, or in the form of plasmid DNA, is introduced into a plant cell. An e:Kample of an AFP-encoding nucleic acid is ~;hown in SEQ ID No. 1. However, the present invention also covers those nucleic acids which differ from the nucleic acid sequence of SEQ ID No. 1 owing to the degeneracy of the genetic code, but which encodes the same AFP amino acid sequence. Moreover, the invention covers thaw mutants or variants of the nucleic acid of SE;Q ID No. 1 which encode an AFP
protein which is active against plant pathogenic fungi of the Ascomycetes family, in particular against Botrytis cinera. 'Ih.ese include, for example, fusion proteins of the AFP ;pratein with other foreign proteins or an AFP protein with an N-terminally deleted methionine. Other examples of variants are nucleic acids which hybridize an the stringent conditions with the nucleic acid of SEQ ID No. 1. The stringent hybridization conditior~s can be determined for example by Sambrook, J. et al. in Molecular Cloning, A
Laboratory Manual, 2nd Edition, Cold Spring Habour Laboratory Press, 1989. Transgenic plants are subsequently regenerated from the transformed plant cells. Preferably, an AFP-encoding nucleic acid can be introduced into the plant cell by means of recombinant agrobacteria, by e:lE>ctroporation, by bombardment with microparticles and/or by means of polyethylene glycol.
The infection with recombinant Agrobacterium tumefaciens bacteria in plant cells of dicots is described, for examp7_e~, by Klee, H. et al.. (1997) Annu.
Rev. Plant Physiol. 38, 467 or EP-B2-0122791). The infection with re:ombinant Agrobacterium tumefaciens bacteria in plant cells of monocots is described, for example, by Ishida, 'Y. et al. (1996) Nature Biotech.
14, 745 with reference to maize (Zea mays L.). The agrobacteria used fcr this purpose have a T-DNA into which the AFP-expressing gene and, if appropriate, a suitable promoter, for example a plant phaseolin promoter (see, for example, EP-B2-0122791) or a viral 35S promoter (see, fcr example, Ishida, Y. et al.
WO 00/229:32 - 5 - PCT/EP99/07364 (1996), above) had been inserted. The T-DNA can be transferred into plant cells for example by coculturing the recornbinant agrobacteria together with immature embryos of the plant in question (see, for example, Ishida, '~. et a1. (1996), above). To select the transformed plant cc=_lls, it is preferred to use T-DNA
construct; which are capable of expressing, in the plant cell, a resistance gene from the AFP gene to be expressed.. A suitable resistance gene is, for example, the gene encoding phosphinothricin acetyltransferase (see, for example, Ishida, Y. et al. (1996}, above).
Thus, successfully transformed plant cells can be selected for example by means of the corresponding antibiotic phosphinothricin. The regeneration of a plant from transformed plant cells is generally known (see, for example, ~~agi et al. (1995), Nature Biotech, 13, 481-485, Ishida, Y. et al. (1996), above, or Schopke et al. (1996;x, Nature Biotech, 14, 731-735.
In addition to the use of Agrobacterium tumefaciens as transformation means, other transformation methods are known and suitable, such as, for example, electro-poration, the bombardment with microparticles or the use of polyethylene glycol (see, for example, Potrykos, (1991) Annu. Rev. Plant Physiol. Mol. Biol., 42, 205;
Estruch, J.J. et al. (1997), Nature Biotech., 15, 137-141 or Dingermann, 'T. (1995) BIOforum, 18, 252). For example, casava (Schopke et al. (1996), above) or banana (Sagi, et a:l. (1995), above) have already been transformed by bombarding plant cells with DNA-coated particles. In this method too, the AFP gene to be expressed is cloned into a suitable vector which preferably has a resistance gene allowing the subsequent selection of the transformed plant cells.
The recombinant erector is preferably applied to tungsten particles with which, for example, suspensions of embryogenic cel~.s are bombarded (see, for example, Sagi, et al. (195), above). The cell suspensions treated thus are sL.bsequently selected for transformed plant ce7.ls using a suitable antibiotic, for example phosphinothricin, and a transgenic plant is regenerated therefrom, for example as already described above.
A considerable advantage of the present invention is that AFP is active against plant pathogenic fungi from the Ascomycetes family, in particular against Botrytis Cinera, which have .already developed a resistance in particular to one or more crop protection agents.
The examples which follow and the figure are intended to describe the invention in greater detail without imposing any limitation:
Sequence description SEQ ID No. 1 shows a nucleic acid sequence encoding an ar~tifungal (223) protein (AFP) from Streptomyces tendae.
Examples Example 1:
To prepare test plates, 50 ~1 of a spore suspension of Botrytis cinerae were added to 100 ml of test agar (20 g/1 malt extra~~t, 10 g/1. glucose, 2 g/1 yeast extract, 0.5 ammor~:ium sulfate, i5 g/1 agar, pH 6.0).
25 ml of this were poured into Petri dishes. The final spore concentratiorv was 105 spores/ml. Sterile anti-biotic test disks (Sc:~leicher and Schuell, Dassel, Frg) 0.5 cm in diameter were subsequently applied to a test plate. Then, 5, 10, 15 and 20 ail of the AFP solution (100 mg/ml lyophili.z~~d culture filtrate of S. tendae-transformants) were subsequently pipetted onto the test disks and the test plate was incubated for 4 days at 30°C. The diameter of the inhibitory zone was 0.7 cm for 5 ~1 of AFP solution, 1 cm diameter for 10 ~1, 1.3 cm for 15 ul ofsolution and 1.9 cm for 20 ~1.
Example 2:
To prepare test plates, 50 ~l of a spore suspension of Botrytis cinerae ZF 3629 (resistant to BCM (Benlat, BenomylTM, Hoechst Sc:~ering AgrEvo GmbH, Frankfurt, carbendazem) were added to 100 ml of test agar (20 g/1 malt extract, 10 g,Jl glucose, 2 g/1 yeast extract, 0.5 ammonium :sulfate, 15 g/1 agar, pH 6.0). 25 ml of this were pou:=ed into Petri dishes. The final spore concentration was :LOS spores/ml. Sterile antibiotic test disks. (Schleic:h~=r and Schuell, Dassel, Frg) 0.5 cm in diameter were swbsequently applied to a test plate.
Then, 5, 10, 15 and 20 ~1 of the AFP solution (100 mg/ml lyophilized culture filtrate of S. tendae-transformants) were subsequently pipetted onto the test disks and the test plate was incubated for 4 days at 30°C. The diameter of the inhibitory zone was 0.9 cm for 5 ~tl of AFP solution, 1.3 cm diameter for 10 ul, 1.9 cm for 15 ~1 of ~~olution and 2.2 cm for 20 ul.
Example 3 - Improved production by fermentation:
In a flask equipped with a hose, 100 ml of the preculture medium (103 g/1 sucrose, 20 g Tryptic Soy Broth (Oxoid, Wesel), 10 g/1 MgCl~ and 10 g/1 yeast extract) were inoculated with an agar section of strain S. tendae. The culture was grown for 5 days at 28°C at 200 rpm on an orbit:~l shaker.
10 1 of the production medium (103 g/1 sucrose, 20 g Tryptic Soy Broth (Uxoid, Wesel), 10 g/1 MgCl2, 10 g/1 yeast extract, 100 ~Cl desmophen, pH 7.5) were inoculated with 7_00 ml of the above-described preculture.
The fermenter employed was a Biostat E (Braun, Melsungenl . Fermentation was carried out for 4 days at 28°C, 700 rpm and 0.5 vvm of air. Thereupon, fermentation was stopped, and the culture supernatant was harvested by cE=_ntrifugation at 3000 rpm and 4°C.
The batch was subsequently dried by lyophilization. It was possible to employ the powder directly. If 15 ~l of the lyophilizate (1.00 mg/ml) were employed against Botrytis cinerae in an inhibitory-zone test, an inhibitory zone of 1.7 cm and, against Botrytis cinerae ZF 3629, an inhibitory zone of 2.3 cm were detected.
Claims (7)
1. The use of an antifungal protein (AFP) having a molecular weight of approx. 10 kDA from Streptomyces tendae against plant pathogenic fungi from the Ascomycetes family, in particular against Botrytis cinera.
2. The use as claimed in claim 1, wherein AFP is used in the form of a formulation together with preferably at least one auxiliary.
3. The use as claimed in claim 1, wherein AFP is formed in a transgenic plant.
4. The use as claimed in claim 3, wherein the transgenic plant is selected from among maize, cotton, potato, banana, Arabidopsis, casava, tobacco, oilseed rape (canola), potato, sugar beet, cereals, strawberries, vegetables, legumes, tomato, lettuce or melon.
5. The use as claimed in claim 4, wherein cereals are selected from amongst wheat, barley or oats, the vegetable used is cabbage, or the legumes are selected from amongst peas or beans.
6. The use as claimed in any of claims 3 - 5, wherein the transgenic plant comprises a nucleic acid as claimed in SEQ ID No. 1, SEQ ID No. 1 being part of the claim.
7. The use as claimed in any of claims 1 - 6, wherein the plant pathogenic fungus stated is resistant to one or more crop protection agents.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19848517A DE19848517A1 (en) | 1998-10-21 | 1998-10-21 | Controlling phytopathogenic fungi, useful particularly against Botrytis cinerea resistant to known fungicides, by treatment with peptide from Streptomyces tendae |
| DE19848517.4 | 1998-10-21 | ||
| PCT/EP1999/007364 WO2000022932A1 (en) | 1998-10-21 | 1999-10-05 | Utilization of an antifungal protein from streptomyces tendae against plant pathogenic fungi |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2347706A1 true CA2347706A1 (en) | 2000-04-27 |
Family
ID=7885179
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002347706A Abandoned CA2347706A1 (en) | 1998-10-21 | 1999-10-05 | Utilization of an antifungal protein from streptomyces tendae against plant pathogenic fungi |
Country Status (14)
| Country | Link |
|---|---|
| EP (1) | EP1123004B1 (en) |
| JP (1) | JP2002527456A (en) |
| KR (1) | KR20010083915A (en) |
| CN (1) | CN1324214A (en) |
| AT (1) | ATE217757T1 (en) |
| AU (1) | AU748455B2 (en) |
| CA (1) | CA2347706A1 (en) |
| DE (2) | DE19848517A1 (en) |
| ID (1) | ID29012A (en) |
| IL (1) | IL142597A0 (en) |
| MX (1) | MXPA01004057A (en) |
| NZ (1) | NZ511213A (en) |
| WO (1) | WO2000022932A1 (en) |
| ZA (1) | ZA200103234B (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100883190B1 (en) * | 2007-08-09 | 2009-02-12 | 경상대학교산학협력단 | Antibiotics preparation containing heat-stable protein having antimicrobial activity |
| CN103708960B (en) * | 2013-12-24 | 2016-06-01 | 安徽师范大学 | A kind of liquid composite microbic bacterial fertilizer and production method and purposes |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2537028A1 (en) * | 1975-08-20 | 1977-03-10 | Bayer Ag | ANTIBIOTICUM, A METHOD FOR ITS MANUFACTURING AND ITS USE AS A PLANT PROTECTION PRODUCT |
| US4158608A (en) * | 1975-08-20 | 1979-06-19 | Bayer Aktiengesellschaft | Fungicidally active antibiotic from Streptomyces tendae Ettlinger |
-
1998
- 1998-10-21 DE DE19848517A patent/DE19848517A1/en not_active Withdrawn
-
1999
- 1999-10-05 IL IL14259799A patent/IL142597A0/en unknown
- 1999-10-05 CA CA002347706A patent/CA2347706A1/en not_active Abandoned
- 1999-10-05 CN CN99812433A patent/CN1324214A/en active Pending
- 1999-10-05 AT AT99952494T patent/ATE217757T1/en not_active IP Right Cessation
- 1999-10-05 EP EP99952494A patent/EP1123004B1/en not_active Expired - Lifetime
- 1999-10-05 JP JP2000576718A patent/JP2002527456A/en active Pending
- 1999-10-05 KR KR1020017004884A patent/KR20010083915A/en not_active Application Discontinuation
- 1999-10-05 WO PCT/EP1999/007364 patent/WO2000022932A1/en not_active Application Discontinuation
- 1999-10-05 DE DE59901521T patent/DE59901521D1/en not_active Expired - Fee Related
- 1999-10-05 ID IDW20010845A patent/ID29012A/en unknown
- 1999-10-05 AU AU64679/99A patent/AU748455B2/en not_active Ceased
- 1999-10-05 MX MXPA01004057A patent/MXPA01004057A/en not_active Application Discontinuation
- 1999-10-05 NZ NZ511213A patent/NZ511213A/en unknown
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2001
- 2001-04-20 ZA ZA200103234A patent/ZA200103234B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| DE19848517A1 (en) | 2000-04-27 |
| AU6467999A (en) | 2000-05-08 |
| EP1123004B1 (en) | 2002-05-22 |
| MXPA01004057A (en) | 2003-03-10 |
| IL142597A0 (en) | 2002-03-10 |
| ZA200103234B (en) | 2002-11-07 |
| AU748455B2 (en) | 2002-06-06 |
| DE59901521D1 (en) | 2002-06-27 |
| WO2000022932A1 (en) | 2000-04-27 |
| EP1123004A1 (en) | 2001-08-16 |
| CN1324214A (en) | 2001-11-28 |
| ID29012A (en) | 2001-07-26 |
| KR20010083915A (en) | 2001-09-03 |
| ATE217757T1 (en) | 2002-06-15 |
| NZ511213A (en) | 2003-03-28 |
| JP2002527456A (en) | 2002-08-27 |
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