U.S. patent application number 11/954367 was filed with the patent office on 2008-04-17 for pseudomycins useful against plant diseases.
Invention is credited to Michael J. Rodriguez, Gary Strobel.
Application Number | 20080090731 11/954367 |
Document ID | / |
Family ID | 22847185 |
Filed Date | 2008-04-17 |
United States Patent
Application |
20080090731 |
Kind Code |
A1 |
Strobel; Gary ; et
al. |
April 17, 2008 |
PSEUDOMYCINS USEFUL AGAINST PLANT DISEASES
Abstract
Plants and crops subject to attack by fungal related diseases
are protected or treated by the application of Pseudomycin
compositions which were originally isolated from Pseudomonas
syringae.
Inventors: |
Strobel; Gary; (Bozeman,
MT) ; Rodriguez; Michael J.; (Indianapolis,
IN) |
Correspondence
Address: |
McDERMOTT, WILL & EMERY
600 13th Street, N.W.
Washington
DC
20005-3096
US
|
Family ID: |
22847185 |
Appl. No.: |
11/954367 |
Filed: |
December 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10343199 |
Aug 18, 2003 |
7326689 |
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PCT/US01/25724 |
Aug 17, 2001 |
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11954367 |
Dec 12, 2007 |
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60226010 |
Aug 18, 2000 |
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Current U.S.
Class: |
504/117 |
Current CPC
Class: |
Y10S 435/822 20130101;
Y10S 435/874 20130101; A01N 63/10 20200101; A01N 43/72 20130101;
A01N 43/713 20130101 |
Class at
Publication: |
504/117 |
International
Class: |
A01N 63/00 20060101
A01N063/00 |
Claims
1-6. (canceled)
7. A method for the protection or treatment of plants and/or crops
against fungal related diseases caused by a fungus selected from
the group consisting of Alternaria helianthi, Aphanomyces sp.,
Drechslera portulacae, Drechslera triciti-repentis, Geotrichim
candidum, Monilinia sp., Mycosphaerella fijienes, Penicillium
roqueforti, Phyllostica maydis, Rhizoctonia solani, Septoria
tritici, Tapesia yallunde, and Ustilago maydis, and imperfect
stages of these fungi, which comprises applying to said plant or
crops an effective amount of a pseudomycin composition.
8. The method of claim 7, wherein said fungus is Alternaria
helianthi, and imperfect stages of this fungus.
9. The method of claim 7, wherein said fungus is Aphanomyces sp.,
and imperfect stages of this fungus.
10. The method of claim 7, wherein said fungus is Drechslera
portulacae, and imperfect stages of this fungus.
11. The method of claim 7, wherein said fungus is Drechslera
triciti-repentis, and imperfect stages of this fungus.
12. The method of claim 7, wherein said fungus is Geotrichim
candidum, and imperfect stages of this fungus.
13. The method of claim 7, wherein said fungus is Monilinia sp.,
and imperfect stages of this fungus.
14. The method of claim 7, wherein said fungus is Mycosphaerella
fijienes, and imperfect stages of this fungus.
15. The method of claim 14, wherein said fungus is Cercospora
sp.
16. The method of claim 7, wherein said fungus is Penicillium
roqueforti, and imperfect stages of this fungus.
17. The method of claim 7, wherein said fungus is Phyllostica
maydis, and imperfect stages of this fungus.
18. The method of claim 7, wherein said fungus is Rhizoctonia
solani, and imperfect stages of this fungus.
19. The method of claim 7, wherein said fungus is Septoria tritici,
and imperfect stages of this fungus.
20. The method of claim 7, wherein said fungus is Tapesia yallunde,
and imperfect stages of this fungus.
21. The method of claim 7, wherein said fungus is Ustilago maydis,
and imperfect stages of this fungus.
22. The method of claim 7, wherein said pseudomycin composition is
isolated from Pseudomonas syringae.
23. The method of claim 7, wherein said pseudomycin composition
comprises an effective amount of molecules selected from the group
consisting of Pseudomycin A, Pseudomycin A', Pseudomycin B,
Pseudomycin B', Pseudomycin C, and Pseudomycin C'.
24. The method of claim 7, wherein said pseudomycin composition is
applied to said plants or crops as an aqueous suspension, solution
or emulsion with a concentration ranging from about 1 to 100
micrograms per mL.
25. The method of claim 7, wherein said plants and/or crops are
selected from the group consisting of bananas, plantains,
sunflower, sugar beets, barley, onion, corn, grapes, portulaca,
wheat, tomato and corn.
26. The method of claim 25, wherein said plants and/or crops are
sugar beets.
27. The method of claim 7, wherein the pseudomycin composition
comprises a molecule comprising the chemical formula I: ##STR2##
wherein R is a lipophilic moiety.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the use of pseudomycins as
effective fungicides against plant and crop diseases, and more
particularly relates to the use of pseudomycins against particular
classes of fungi which cause diseases in plants and crops.
BACKGROUND
[0002] One class of new antifungal agents, the pseudomycins, shows
great promise for treating fungal infections in a variety of
patients. (see i.e., Harrison, L., et al., "Pseudomycins, a family
of novel peptides from Pseudomonas syringae possessing
broad-spectrum antifungal activity," J. Gen. Microbiology, 137(12),
2857-65 (1991) and U.S. Pat. Nos. 5,576,298 and 5,837,685).
Pseudomycins are natural products derived from isolates of
Pseudomonas syringae. P. syringae is a large group of
plant-associated bacteria that have been the source of several
bioactive substances, such as bacitracin and the syringomycins.
Natural strains and transposon-generated mutants of P. syringae
produce compounds with antifungal activity. A transposon-generated
regulatory mutant of the wild type strain of P. syringae MSU 174,
known as MSU 16H (ATCC 67028), produces several pseudomycins.
Pseudomycins A, B, C and C' have been isolated, chemically
characterized, and shown to possess wide spectrum antifungal
activity, including activity against important fungal pathogens in
both humans and plants. The pseudomycins are structurally related
to but are distinct from syringomycin and other antimycotics from
isolates of P. syringae. The peptide moiety for pseudomycins A, B,
C, C' corresponds to
L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp(3-OH)-L-Thr(4-Cl)
with the terminal carboxyl group closing a macrocyclic ring on the
OH group of the N-terminal Ser. The analogs are distinguished by
the N-acyl side chain, i.e., pseudomycin A is N-acylated by
3,4-dihydroxytetradeconoate, pseudomycin B by
3-hydroxytetradecanoate, pseudomycin C by
3,4-dihydroxyhexadecanoate and pseudomycin C' by
3-hydroxyhexadecanoate. (see i.e., Ballio, A., et al., "Novel
bioactive lipodepsipeptides from Pseudomonas syringae: the
pseudomycins," FEBS Letters, 355(1), 96-100, (1994) and Coiro, V.
M., et al., "Solution conformation of the Pseudomonas syringae MSU
16H phytotoxic lipodepsipeptide Pseudomycin A determined by
computer simulations using distance geometry and molecular dynamics
from NMR data," Eur. J. Biochem., 257(2), 449-456 (1998).)
[0003] The present invention provides a group of pseudomycins which
are particularly useful to protect plants and crops against fungal
diseases.
SUMMARY OF THE INVENTION
[0004] It is an object of the present invention to provide a method
for the treatment or protection of plants and crops against
diseases.
[0005] A further object of the invention is to provide a method for
the treatment or protection of plants and crops by application of
certain pseudomycins.
[0006] An even further object of the invention is the use of
certain pseudomycins to protect or treat plants and crops against
diseases caused by fungi.
[0007] Other objects and advantages of the invention will become
apparent as the description of the invention proceeds.
[0008] In satisfaction of the foregoing objects and advantages, the
present invention provides a method for the protection or treatment
of plant and crops against fungal-related diseases, which comprises
applying to said plants or crops an effective amount of one or more
pseudomycin products.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0009] The present disclosure relates to the discovery of novel,
previously unsuspected uses for the class of lipopeptides known
collectively as the pseudomycins as fungicides or antimycotic
agents. In a preferred embodiment, the pseudomycins, are
individually and as a group, particularly useful in the treatment
or protection of plants challenged by a group of Ascomyceteous
fungi related to Mycosphaerella asp. (perfect or sexual stage of
the fungus) and virtually all of the various imperfect stages of
this fungus that are known, including Septoria sp., and Cercospora
sp. In addition, a number of other extremely economically important
plant pathogenic fungi are killed by the pseudomycins including
Tapesia yallundae, Ustilago maydis, Penicillum roqueforti,
Monilinia sp. and Geotrichum candidum. Thus, the pseudomycins,
alone or individually, have use in treating plants to protect them
from harm caused by these fungi.
[0010] The types of diseases caused by these organisms range from
plants in storage (fruits and vegetables) to extremely important
field diseases such as Black Sigatoka of banana and straw breaker
and blotch of wheat.
[0011] This discovery relates to a previously unsuspected set of
extremely important plant pathogenic fungi that seem to be
biologically related and are sensitive to one or more of the
pseudomycins and are both inhibited and killed by them. These fungi
and a few others, not previously disclosed, cause some of the most
important plant diseases on the planet. Currently these
fungal-caused diseases are being controlled by simple or more
complex mixtures of man-made fungicides which cause environmental
damage and may be an unsuspected threat to human health. The
pseudomycins, on the other hand, offer a safe, and effective
alternative to the use of man-made chemicals for the control of
certain plant diseases. In addition, the use of the pseudomycins
for plant disease control have certain benefits since the use of
natural products for disease control would allow the producer to
proclaim that the crop has been grown under biological/organic
conditions allowing for a higher profit to be made on the product.
This is noteworthy since no major crop in the world currently has
applied to it any naturally produced fungicide for plant disease
control. The pseudomycins certainly offer many benefits to both the
agricultural producer as well as the consumer.
[0012] As an example of how and why the pseudomycins may be useful
to the world's agriculture, the minimum inhibitory concentrations
(MICs) for several of the pseudomycins e.g. Pseudo A, B, B'C, and
C' are in the range of 1 mg or less per ml. This is an extremely
desirable concentration for effective application in agricultural
situations. These compounds produce an even greater effect (less
than 1.0 mg) when tested against M. fijiensis isolate 8088/88. M,
fijensis is the causal organism of the Black Sigatoka disease of
bananas and plantains. Currently, the producers of these crops,
worldwide, must spray a mixture of three fungicides (man-made) at
the rate of 30 times per year in order to have a banana crop. This
one disease alone represents the largest consumption of fungicide
per crop in the entire world. The disadvantages for the use of
these synthetic fungicides are numerous including: 1. their
extremely high cost ($ millions); 2. the inability of the producer
to sell organically grown produce since fungicides have been
applied to it; and 3. the uncertainty to human as well the
environmental health risks involved in the continuous use of the
fungicides over decades. The soil beneath the banana canopy in the
plantations appears sterile of animal life and shows a build up of
fungicide residues. On the other hand, the naturally produced
pseudomycins appear more effective in controlling the sigatoka
disease, while at the same time offering benefits to the
environment and to human health.
[0013] In addition, the pseudomycins are effective against a number
of other plant disease causing fingi including the fungi that
destroy plant produce in storage e.g. Penicillium sp., Monilinia
sp. and Geotrichum sp. A mixture of pseudomycins applied to
harvested fruit would preclude fungal infections and storage
rots.
[0014] Still other possibilities for the applications of the
pseudomycins include applications for the control of diseases
caused by Septoria sp., specifically S. nodurum and S. triticii on
wheat, but also, based on the biological activity of these
molecules-virtually any Septoria sp. causing any plant disease in
the world. Likewise, other fungi related to Mycosphaeella sp. are
affected and they include plant diseases caused by Cercospora sp.
which causes leaf spot of sugar beets and many other crops. Other
disease causing organisms are also affected by the pseudomycins and
they include Dreschslera portulaceae.
[0015] In accordance with the present invention it has been
discovered that the pseudomycins described herein possess enormous
antimycotic activity against a previously unsuspected, and closely
related group of plant pathogenic fungi. This main group is
represented by the perfect stage fungus sp. Mycosphaeella sp. and
each of its representatives in the imperfect stage (asexual stage)
such as Septoria sp. and Cercospora sp. Generally, the pseudomycins
may be used alone or as a mixture in a formulation to protect
plants from fungal infection. Applications to crops in the field as
well as in storage are visualized as the potential uses of these
compounds.
[0016] Thus, it is a purpose of this invention to demonstrate that
a number of extremely economically important plant pathogenic fungi
are susceptible to the effects of one or more pseudomycins which
were originally isolated from the plant associated
bacterium-Pseudomonas syringae.
[0017] The pseudomycins useful in the method of the present
invention are preferably pseudomycins produced by the Pseudomonas
syringae including the pseudomycins identified as Pseudomycins A,
A', B, B', C and C' as well as derivatives such as pseudomycin
A-PO4, a phosphate derivative and pseudomycin A-FB, both of which
are known.
[0018] These pseudomycins are applied against a wide variety of
plants and crops which are susceptible to parasitic diseases caused
by fungi. In that connection, the pseudomycin compositions of the
present invention are primarily useful to prevent the onset of
parasitic diseases caused by fungi so that treatment of the plants
and crops prior to onset of the disease is preferred. However, the
pseudomycin compositions are also useful in treatment of infected
plants.
[0019] Pseudomycin compositions of the present invention are
effective at very low concentrations on the order of 1 up to 100
micrograms of pseudomycin per ml of aqueous solution. In that
regard, a preferred method of application of the pseudomycin
composition of this invention is by treatment as by spraying
directly onto the plant or crop to be treated using the indicated
concentrations. The pseudomycin compositions of the present
invention may be in the form of solutions, suspensions or
emulsions, or any other form suitable for spraying onto the plants
and crops.
[0020] The preferred pseudomycins used in the present invention and
their methods of preparation are known or are fully disclosed and
described in copending applications PCT/US00/08728, filed Apr. 14,
2000 and PCT/US00/08727, filed Apr. 14, 2000 both applications
designating the United States. The disclosures of both these
applications are incorporated herein by reference in their
entirety.
[0021] As used herein, the term "pseudomycin" refers to compounds
having the following formula I: ##STR1## where R is a lipophilic
moiety. The pseudomycin compounds A, A', B, B', C, C' are
represented by the formula I above where R is as defined below.
[0022] Pseudomycin A R=3,4-dihydroxytetradecanoyl
[0023] Pseudomycin A' R=3,4-dihydroxypentadecanoate,
[0024] Pseudomycin B R=3-hydroxytetradecanoyl
[0025] Pseudomycin B' R=3-hydroxydodecanoate
[0026] Pseudomycin C R=3,4-dihydroxyhexadecanoyl
[0027] Pseudomycin C' R=3-hydroxyhexadecanoyl
[0028] As used herein, pseudomycin refers to one or more members of
a family of antifungal agents that has been isolated from the
bacterium Pseudomonas syringae. A pseudomycin is a
lipodepsipeptide, a cyclic peptide including one or more unusual
amino acids and having one or more appended hydrophobic or fatty
acid side chains. Specifically, the pseudomycins are
lipodepsinonapeptides, with a cyclic peptide portion closed by a
lactone bond and including the unusual amino acids
4-chlorothreonine, 3-hydroxyaspartic acid, dehydro-2-aminobutyric
acid, and 2,4-diaminobutyric acid. It is believed that these
unusual amino acids are involved in biological characteristics of
the pseudomycins, such as stability in serum and their killing
action. Pseudomycins include pseudomycin A, pseudomycin A',
pseudomycin B, pseudomycin B', pseudomycin C, and pseudomycin C'.
Each of these pseudomycins has the same cyclic peptide nucleus, but
they differ in the hydrophobic side chain attached to this
nucleus.
[0029] Pseudomycins A, A', B, B', C and C' have each been isolated
and purified and their structures have been characterized by
methods including amino acid sequencing, NMR, and mass
spectrometry. Pseudomycins A, B, C, and C' are discussed in U.S.
Pat. No. 5,576,298, issued Nov. 19, 1996 to G. Strobel et al.;
Harrison et al., "Pseudomycins, a family of novel peptides from
Pseudomonas syringae possessing broad-spectrum antifungal
activity," J. Gen. Microbiology 137, 2857-2865 (1991); and Ballio
et al., "Novel bioactive lipodepsipeptides from Pseudomonas
syringae: the pseudomycins," FEBS Lett. 355, 96-100 (1994).
Pseudomycins A' and B' are described in U.S. Patent Application
Serial No. PCT/US00/08727, by Palaniappan Kulanthaivel, et al.
entitled "Pseudomycin Natural Products" submitted even date
herewith and exemplified in the Examples, and incorporated herein
by reference in its entirety. Antifungal activity due to several
pseudomycins was traced to P. syringae bearing a transposon known
as Tn 903, which encodes factors including kanamycin resistance.
The sequence of and methods for manipulating transposon Tn 903 are
known. Oka et al., "Nucleotide sequence of the kanamycin resistance
transposon Tn 903," J. Mol. Biol. 147, 217-226 (1981). Each of the
references cited in this paragraph is specifically incorporated
herein by reference.
[0030] The pseudomycins vary in structure and properties. Preferred
pseudomycins A, B, C and C' exhibit activity against a wide variety
of fungi and also exhibit generally acceptable toxicity. Compared
to the other preferred pseudomycins, pseudomycin B has greater
potency against certain fungi and a lower level of toxicity.
Therefore, for the present methods, pseudomycin B is more
preferred. Each pseudomycin has a cyclic nonapeptide ring having
the sequence Ser-Dab-Asp-Lys-Dab-aThr-Dhb-HOAsp-CIThr (Serine;
2,4-Diaminobutyric acid; Aspartic acid; Lysine; 2,4-Diaminobutyric
acid; alloThreonine; Dehydro-2-aminobutyric acid; 3-hydroxyAspartic
acid; 4-chloroTheonine), more specifically,
L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp(3-OH)-L-Thr(4-Cl),
with the carboxyl group of the ClThr and the hydroxyl group of the
serine closing the ring with a lactone bond. The pseudomycins
differ in the nature of the lipophilic moiety that is attached to
the amine group of the N-terminal serine. The amine group of the
serine forms an amide bond with the carboxyl of a
3,4-dihydroxytetradecanoyl moiety in pseudomycin A, a
3-monohydroxytetradecanoyl moiety in pseudomycin B, a
3,4-dihydroxyhexadecanoyl moiety in pseudomycin C and a
3-monohydroxyhexadecanoyl moiety in pseudomycin C'. The carboxyl
group of the serine forms an amide bond with the Dab of the
ring.
[0031] The pseudomycins used in the present invention may be used
as their acceptable salts. The term "acceptable salt", as used
herein, refers to salts of the compounds described above that are
substantially non-toxic to living organisms. Typical acceptable
salts include those salts prepared by reaction of the compounds of
the present invention with a mineral or organic acid or an
inorganic base. Such salts are known as acid addition and base
addition salts.
[0032] Acids commonly employed to form acid addition salts are
mineral acids such as hydrochloric acid, hydrobromic acid,
hydroiodic acid, sulfuric acid, and phosphoric acid, and organic
acids such as p-toluenesulfonic, methanesulfonic acid, oxalic acid,
p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric
acid, benzoic acid, and acetic acid. Examples of such
pharmaceutically acceptable salts are the sulfate, pyrosulfate,
bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,
dihydrogenphosphate, metaphosphate, pyrophosphate, chloride,
bromide, iodide, acetate, propionate, decanoate, caprylate,
acrylate, formate, isobutyrate, caproate, heptanoate, propiolate,
oxalate, malonate, succinate, suberate, sebacate, fumarate,
maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate,
chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate,
methoxybenzoate, phthalate, sulfonate, xylenesulfonate,
phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,
gamma-hydroxybutyrate, glycollate, tartrate, methanesulfonate,
propanesulfonate, naphthalene-1-sulfonate,
napththalene-2-sulfonate, and mandelate. Preferred pharmaceutically
acceptable acid addition salts are those formed with mineral acids
such as hydrochloric acid and hydrobromic acid, and those formed
with organic acids such as maleic acid and methanesulfonic
acid.
[0033] Base addition salts include those derived from inorganic
bases, such as ammonium or alkali or alkaline earth metal
hydroxides, carbonates, and bicarbonates. Such bases useful in
preparing the salts of this invention thus include sodium
hydroxide, potassium hydroxide, ammonium hydroxide, potassium
carbonate, sodium carbonate, sodium bicarbonate, potassium
bicarbonate, calcium hydroxide, and calcium carbonate. The
potassium and sodium salt forms are particularly preferred.
[0034] It should be recognized that the particular counterion
forming a part of any salt of this invention is not of a critical
nature, so long as the salt as a whole is pharmacologically
acceptable and as long as the counterion does not contribute
undesired qualities to the salt as a whole.
[0035] The present invention may be better understood with
reference to the following examples. These examples are intended to
be representative of specific embodiments of the invention, and are
not intended as limiting the scope of the invention.
Biological Materials on Deposit
[0036] P. syringae MSU 16H is publicly available from the American
Type Culture Collection, Parklawn Drive, Rockville, Md., USA as
Accession No. ATCC 67028. P. syringae strains 25-B1, 7H9-1, and 67
H1 were deposited with the American Type Culture Collection on Mar.
23, 2000 and were assigned the following Accession Nos.:
[0037] 25-B1 Accession No. PTA-1622
[0038] 7H9-1 Accession No. PTA-1623
[0039] 67 H1 Accession No. PTA-1621
[0040] The pseudomycins were isolated from liquid cultures of
Pseudomonas syringae. Pseudomonas syringae is a plant-associated
microbe producing a variety of phytotoxins and other complex
peptides 1-3. In the late 1980s, it was shown that P. syringae was
producing antifungal agents. Basically, the concept that
endosymbionts growing on the plant produce antifungal agents to
protect the plant from fungal diseases. The pseudomycins were
identified as the bioactive antifungal agents. A
transposon-generated mutant of P. syringae wild type was shown to
be hyper-producers of these natural products. These transposon
mutants strains.sup.4 developed at Montana State University were
used successfully to inoculate elm trees to control Dutch Elm
Disease.sup.5-6. In addition, these natural products have shown
selective antifungal activity against diseases found on field
crops, fruits and other plants (Tables 1-3). For example, the
pseudomycins shows promising activity against M. fijiensis (M.
fijiensis causing black sigatoka of bananas requires more fungicide
and fungicide applications than any other plant disease in the
world today. The pseudomycins were also shown to prevent premature
spoilage of mangoes.
Example 1
[0041] Reproducible large-scale production (kilograms) of these
natural products has been successfully demonstrated. Purified
samples of free base and alternative salt forms were prepared.
Approximately, 34 fungal plant pathogens were used to evaluate the
antifungal properties of pseudomycin A, B, B', C, C'. Inhibitory
concentrations were measured at two-time points day 2 and 5.
TABLE-US-00001 TABLE 1 Antifungal Activity tested with pathogens of
interest: a. Alternaria helianthi = leaf spot of sunflower b.
Aphanomyces sp. = root rot, seedling wilt of many plants including
sugarbeets c. Bipolaris sorokiniana = kernal blight of barley d.
Botrytis alli = gray mold neck rot of onion e. Cochliobolis
carbonum = leaf blight of corn f. Diplodia natalensis = bunch rot
of grapes g. Dreschslera portulacae = leaf spot of portulacs h. D.
teres = barley net blotch i. D. tritici-repentis leaf spot of wheat
j. Fusarium avenaceum = root rot of several field crops k. F.
culmorum = fusarium blight or scab l. F. oxysporum = fusarium
vascular wilt m. F. solani = root rot n. Geotrichum candidum =
tomato field rot o. Monilinia fructicola = brown rot of stone
fruits p. Mycosphaeella fijiensis = black sigatoka of banana (FIG.
3) q. Penicillum roqueforti = green mold of fruit in storage r.
Phyllosticta maydis = yellow leaf spot of corn s. Phytophthoras
causing blight, rots decay t. Rhizoctonia solani = rhizoctonia root
rot u. Sclerotina sclerotiorum = crown rot of many plant species v.
Septoria tritici = leaf blotch and glume blotch of wheat (FIG. 5)
w. Tapesia acuformis = take all disease of wheat X. Ustilago maydis
= smut of corn y. Verticillum dahlia = verticillum wilt of many
crops and tree species. =Very sensitive towards pseudomycins
Materials and Methods
[0042] Fungi to be tested were propagated on potato dextrose agar
(PDA) plates at room temperature. Stock solutions of pseudomycins
and depsipeptide were suspended in dimethylsulfoxide (DMSO) (Sigma)
at 5 mg/ml and stored at -20 C. Serial dilutions of compounds were
done the day of the experiment. All serial dilutions were made in
DMSO. Note: an initial experiment of pseudomycins B and B' in
methanol was performed on some of the fungi, and several fungi
showed inhibited growth in the presence of methanol. These are
noted in the raw data section.
[0043] Assays were performed in 24-well cell culture clusters
(Costar 3524), with 990 ul potato dextrose broth (PDB, Difco) and
10 ul of the compound to be tested in each well. Initial
concentrations tested were 50-1.56 ug/ml (final concentration of
pseudomycin). Actual concentrations of pseudomycins tested were 50,
25, 12.5, 6.25, 3.12 and 1.56 ug/ml. Each well was inoculated with
the appropriate fungus. Unless noted, the fungal inoculum consisted
of an .about.4 mm.sup.2 piece of PDA with fungal mycelium. The
exceptions were as follows. Ustilago maydis: this fungus grows much
like a yeast on PDA, and cells were scraped from the PDA stock
plate, resuspended in PDB and 10 ul inoculum added to each well.
Monilinia sp.: this fungal mycelium grows as a very loose sheet on
top of PDA and would not adhere to agar blocks. Fungal mycelium was
ground with a metal rod in 500 ul PDB and 10 ul inoculum was added
to each well. Mycosphaeella fijiensis, Septoria passerinii,
Septoria triticii: these fungi all grow very slowly and the results
were difficult to score if a small piece of agar with fungal
mycelium on it was used as an inoculum. For these fungi, the
mycelium was ground with a metal rod in 500 ul PDB and 10 ul
inoculum was added to each well.
[0044] Wells were scored for fungal growth at two days and five
days. Some slow growing fungi were scored at later days for growth
because assessment was not possible at two or five days. Growth was
scored by comparison to a control consisting of fungus inoculated
into 990 ul potato dextrose broth and 10 ul of DMSO
(Dimethylsulfoxide). An additional control consisting of fungal
inoculum in PDB only was performed to ensure that the DMSO was not
inhibitory to fungal growth. DMSO did not affect any of the fungi
tested, with the exception of Drechslera portulacae, where some
inhibition was noted.
[0045] Any fungi that showed inhibition were retested. For assays
that were repeated, new stock solutions were made from a second
shipment of pseudomycins. Note that pseudomycin B' and depsipeptide
stock solutions were remade from first shipment materials because
these were not included in the second shipment. Several of these
fungi showed no growth even at the lowest levels of pseudomycins
added, and were retested a third time using lower levels of
pseudomycins (2-0.0625 ug/ml final concentration of pseudomycin).
Actual concentrations of pseudomycins tested were 2.1, 0.5, 0.25,
0.125 and 0.0625 ug/ml. A-PO.sub.4 and A-FB pseudomycin A phosphate
salt and free base respectively. TABLE-US-00002 TABLE 2 Two day
results - concentration indicated is lowest level of compound
(ug/ml) that results in no growth. NI, no inhibition; p (partial),
at least 50% inhibited; --, not determined. If number of assays was
greater than one, number of assays performed on the fungus is noted
in parentheses. For several slow-growing fungi, days elapsed before
observation (if different than two) are noted. Pseudomycin-
A-PO.sub.4 A-FB B B' C C' Alternaria helianthi (2.times.) 25 25 NI
25 p at 50 p at 50 Aphanomyces sp. (2.times.) 50 NI p at 50 25 25
25 Bipolaris sorokiniana NI NI NI NI NI NI Botrytis alli (2.times.)
50 NI NI NI NI NI Cochliobolus carbonum NI NI NI NI NI NI Diplodia
natalensis NI NI NI NI NI NI Drechslera portulacae (3.times.) 0.06
0.25 0.06 0.12 0.06 0.05 Drechslera teres NI NI NI NI NI NI
Drechslera tritici-repentis (2.times.) 50 25 50 50 50 50 Fusarium
avenaceum NI NI NI NI NI NI Fusarium culmorum NI NI NI NI NI NI
Fusarium oxysporum cubense NI NI NI NI NI NI Fusarium solani NI p
at 50 p at 50 NI p at 50 NI Geotrichim candidum (3.times.) 12.5
3.12 1.56 25 3.12 1.56 Monilinia sp. (2.times.) 12.5 6.25 3.13 25
6.25 3.12 Mycosphaeella fijiensis (Sigatoka) (7 day, 1 0.5 0.5 0.5
1.56 1.56 3.times.) Mycosphaeella fijiensis (8088/88) (7 day, 0.12
0.25 0.06 0.5 0.125 0.125 3.times.) Penicillium roqueforti
(3.times.) 0.5 1 0.5 1.56 1.56 1.56 Pestalotiopsis microspora NE-32
p at 50 p at 50 p at 50 NI NI NI Phoma chrysamthecola -- -- -- --
-- -- Phyllosticta maydis p at 50 p at 50 50 p at 50 p at 50 p at
50 Phytophthora cactorum NI NI NI NI NI NI Phytophthora cinnemani
NI NI NI NI NI NI Phytophthora parasitica NI NI NI NI NI NI
Phytophthora ultimum NI NI NI NI NI NI Rhizoctonia solani
(2.times.) 6.25 6.25 6.25 6.25 25 p at 50 Sclerotinia sclerotiorum
(2.times.) NI NI p at 50 NI NI 50 Septoria passerinii (3.times.)
0.125 0.06 0.06 0.5 0.06 0.06 Septoria tritici (3.times.) 0.25 0.25
0.06 0.5 0.125 0.125 Stagonospora nodorum NI NI NI NI NI NI Tapesia
acuformis (2.times.) 50 NI NI NI sol NI Tapesia yallundae
(2.times.) 25 12.5 25 6.25 25 6.25 Ustilago maydis (3.times.) 0.5 1
0.5 1.56 0.25 0.25 Verticillium dahliae (2.times.) p at 50 50 25 NI
25 p at 50
[0046] TABLE-US-00003 TABLE 3 Five day results - concentration
indicated is lowest level of compound (ug/ml) that results in no
growth. NI, no inhibition; p (partial), at least 50% inhibited; --,
not determined (slow growth). If number of assays was greater than
one, number of assays performed on the fungus is noted in
parentheses. For several slow-growing fungi, days elapsed before
observation (if different than five) are noted. Pseudomycin-
A-PO.sub.4 A-FB B B' C C' Alternaria helianthi (2.times.) 25 25 50
25 NI NI Aphanomyces sp. (2.times.) p at 50 NI 50 NI 50 p at 50
Bipolaris sorokiniana NI NI NI NI NI NI Botrytis alli (2.times.) p
at 50 NI NI NI NI NI Cochliobolus carbonum NI NI NI NI NI NI
Diplodia natalensis NI NI NI NI NI NI Drechslera portulacae
(3.times., 9 day) 0.06 0.25 0.06 0.25 0.06 0.5 Drechslera teres NI
NI NI NI NI NI Drechslera tritici-repentis p at 50 NI p at 50 p at
50 NI NI Fusarium avenaceum NI NI NI NI NI NI Fusarium culmorum NI
NI NI NI NI NI Fusarium oxysporum cubense NI NI NI NI NI NI
Fusarium solani NI NI NI NI NI NI Geotrichim candidum (3.times.)
6.25 6.25 3.12 25 25 6.25 Monilinia sp. (2.times.) 12.5 12.5 6.25
25 12.5 6.25 Mycosphaeella fijiensis (Sigatoka) (3.times., 1 1 1 1
1.56 1.56 21 days) Mycosphaeella fijiensis (8088/88) (3.times.,
0.25 0.25 0.12 1 0.25 0.25 21 days) Penicillium roqueforti
(3.times.) 3.12 3.12 6.25 1.56 12.5 p at 50 Pestalotiopsis
microspora NE-32 NI NI p at 50 NI NI NI Phoma chrysamthecola NI p
at 50 p at 50 p at 50 NI NI Phyllosticta maydis NI NI 50 NI NI NI
Phytophthora parasitica NI NI NI NI NI NI Phytophthora cinnemani NI
NI NI NI NI NI Phytophthora ultimum NI NI NI NI NI NI Phytophthora
cactorum NI NI NI NI NI NI Rhizoctonia solani (2.times.) 12.5 50 50
6.25 p at 50 p at 50 Sclerotinia sclerotiorum (2.times.) NI NI NI
NI NI p at 50 Septoria tritici (3.times.) 0.25 0.25 0.06 0.5 0.25
0.25 Septoria passerinii (3.times.) 0.12 0.06 0.06 0.5 0.06 0.06
Stagonospora nodorum NI MI NI NI NI NI Tapesia acuformis (2.times.)
50 NI NI NI NI NI Tapesia yallundae (2.times.) 50 25 NI 50 NI NI
Ustilago maydis (3.times.) 1 1 0.5 3.12 0.25 0.25 Verticillium
dahliae (2.times.) p at 50 p at 50 p at 50 NI p at 50 p at 50
[0047] A review of the above experiments demonstrates that the
fungi tested were best inhibited by one or several of the
pseudomycins, rather than responding in the same manner to all of
the compounds tested. Six fungi that showed no growth even at the
lowest concentration of pseudomycins initially tested, 1.56 ug/ml.
These fingi were retested with even lower concentrations of
pseudomycins. E.g. Drechslera portulacae appeared to have no growth
even at 0.0625 ug/ml pseudomycin A (PO.sub.4), B and C (9 days).
Two different isolates of Mycosphaeella fijiensis responded
differently to the lower doses of pseudomycins. The Sigatoka
isolate appeared to be inhibited by each of the pseudomycins down
to .about.1 ug/ml. However, 8088/88 isolate was best inhibited by
pseudomycin B, with no growth at 0.125 ug/ml at 21 days. Septoria
tritici and Septoria passerinii were strongly inhibited by all the
pseudomycins, with S. tritici showing no growth at 5 days at 0.0625
ug/ml pseudomycin B, and S. passerinii showing no growth at 5 days
at 0.0625 ug/ml pseudomycin A (free base), B, C and C'. Ustilago
maydis was best inhibited by either pseudomycin C or C', with no
growth at 0.25 ug/ml at 5 days. Some of these fungi showed only
minor growth inhibition at the highest levels of pseudomycins
tested (e.g. Alternaria helianthi, Aphanomyces sp., Botrytis alli,
Sclerotinia sclerotiorum, Tapesia acuformis, Tapesia yallundae and
Verticillium dahliae). Several fungi showed good inhibition with
one or several pseudomycins, but not all. These include Rhizoctonia
solani, which was best inhibited by B' (no growth at 6.25 ug/ml at
5 days), Monilinia sp., best inhibited by B and C' (no growth at
6.25 ug/ml at 5 days), Geotrichim candidum, best inhibited by B (no
growth at 3.12 ug/ml at 5 days) and Penicillium roqueforti, best
inhibited by B' (no growth at 1.56 ug/ml at 5 days).
[0048] From this data, it can be concluded that the pseudomycins
are a group of selective "natural" fungicides. Some fungi causing
infections found in post harvest crops and other plant species are
sensitive to the pseudomycins (e.g. Penicillium and Geotrichum).
Pseudomycin shows impressive activity against M. fijiensis
(bananas). Crude preps as well as purified materials have a
potential role in plant disease control. Large scale production of
the pseudomycins is feasible and relatively inexpensive to produce.
Natural products are likely to be environmentally compatible and
potentially safe
REFERENCES
[0049] 1. A. Ballio, F. Bossa, D. DiGiorgio, P. Ferranti, M. Paci,
P. Pucci, A. Scaloni, A. Segre, G. A. Strobel. FEBS Letters, 1994,
355, 96-100. [0050] 2. C. Potera. Science, 1994, 265, 605. [0051]
3. L. Harrision, D. B. Teplow, M. Rinaldi, G. A. Strobel. J.
General Microbiology 1991, 137, 2857-2865. [0052] 4. B. Lam, G.
Strobel, L. Harrison, S. Lam. Proc. Natl. Acad. Sci. 1987, 84,
6447-6451. [0053] 5. R. Scheffer, D. Elgersma, G. Strobel. Neth. J.
Pl. Path. 1989, 95, 293-304. [0054] 6. R. Scheffer, D. Elgersma, L.
A. DeWeger, G. Strobel. Neth. J. Pl. Path. 1989, 95, 281-292.
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