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..
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 US Patent 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 thePseudomonas 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).)
The present invention provides a group of pseudomycins which are particularly
useful to protect plants and crops against fungal diseases.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for the
treatment or protection of plants and crops against diseases.
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.
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.
Other objects and advantages of the invention will become apparent
as the description of the invention proceeds.
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
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 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 includingTapesia 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.
The types of diseases caused by these organisms range from plants
in storage (Suits and vegetables) to extremely important field diseases such as
Black Sigatoka of banana and straw breaker and blotch of wheat.
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.
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, A',B,B'C, and C' are in the range of 1mg 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
1.0mg) when tested against M. fijiensis isolate 8088/88. M. fijiensis
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.
In addition, the pseudomycins are effective against a number of other
plant disease causing fungi 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
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
Mycosphaerella sp. are affected and they include plant diseases caused byCercospora
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
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 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.
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.
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.
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.
Pseudomycin compositions of the present invention are effective at
very low concentrations on the order of 1 up to 100 µgrams 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.
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 April 14,2000 and PCT/US00/08727, filed April
14,2000 both applications designating the United States.
As used herein, the term "pseudomycin" refers to compounds having
the following formula I:
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.
Pseudomycin AR = 3,4-dihydroxytetradecanoylPseudomycin A'R = 3,4-dihydroxypentadecanoate,Pseudomycin BR = 3-hydroxytetradecanoylPseudomycin B'R = 3-hydroxydodecanoatePseudomycin CR = 3,4-dihydroxyhexadecanoylPseudomycin C'R = 3-hydroxyhexadecanoyl
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.
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. Patent
No. 5,576,298, issued November 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. 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).
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-ClThr
(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.
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.
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
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.
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.
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
Biological Materials on Deposit
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 March 23, 2000 and were assigned the following
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
strains4 developed at Montana State University were used successfully
to inoculate elm trees to control Dutch Elm Disease5-6. 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.
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.
Antifungal Activity tested with pathogens of interest:a.Alternaria helianthi = leaf spot of sunflowerb.Aphanomyces sp.= root rot, seedling wilt of many plants
including sugarbeetsc.Bipolaris sorokiniana = kernal blight of barleyd.Botrytis alli = gray mold neck rot of onione.Cochliobolis carbonum = leaf blight of cornfDiplodia natalensis = bunch rot of grapesg.Dreschslera portulacae = leaf spot of portulacs√h.D. teres = barley net blotchi.D. tritici-repentis leaf spot of wheatj.Fusarium avenaceum = root rot of several field cropsk.F. culmorum = fusarium blight or scabl.F. oxysporum = fusarium vascular wiltm.F. solani = root rotn.Geotrichum candidum = tomato field rot√o.Monilinia fructicola = brown rot of stone fruitsp.Mycosphaeella fijiensis = black sigatoka of banana√
(Figure 3)q.Penicillum rogueforti = green mold of fruit in storage√r.Phyllosticta maydis = yellow leaf spot of corns.Phytophthoras causing blight, rots decayt.Rhizoctonia solani = rhizoctonia root rotu.Sclerotina sclerotiorum = crown rot of many plant speciesv.Septoria tritici = leaf blotch and glume blotch of wheat√
(Figure 5)w.Tapesia acuformis = take all disease of wheatx.Ustilago maydis = smut of corn√y.Verticillum dahlia = verticillum wilt of many crops and
tree species.= Verysensitive towards pseudomycins
Materials and Methods
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.
Assays were performed in 24-well cell culture clusters (Costar 3524),
with 990 ul potato dextrose broth (PDB, Difco) and 10µl of the compound to be tested
in each well. Initial concentrations tested were 50 - 1.56 µg/ml (final concentration
of pseudomycin). Actual concentrations of pseudomycins tested were 50, 25, 12.5,
6.25, 3.12 and 1.56 µg/ml. Each well was inoculated with the appropriate fungus.
Unless noted, the fungal inoculum consisted of an ∼ 4 mm2 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 µl 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 µl PDB and 10µl inoculum was added to each well. Mycosphaerella 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 500µl PDB and 10 µl inoculum was added to each well.
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 µl potato dextrose broth and 10 µl 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.
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 µg/ml
final concentration of pseudomycin). Actual concentrations of pseudomycins tested
were 2,1, 0.5, 0.25, 0.125 and 0.0625 µg/ml. A-PO4 and A-FB = pseudomycin
A phosphate salt and free base respectively.
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-PO4A-FBBB'CC'Alternaria helianthi (2x)2525NI25p at 50p at 50Aphanomyces sp. (2x)50NIp at 50252525Bipolaris sorokinianaNINININININIBotrytis alli (2x)50NININININICochliobolus carbonumNINININININIDiplodia natalensisNINININININIDrechslera portulacae (3x)0.060.250.060.120.060.05Drechslera teresNINININININIDrechslera tritici-repentis (2x)502550505050Fusarium avetiaceumNINININININIFusarium culmorumNINININININIFusarium oxysporum cubenseNINININININIFusarium solaniNIp at 50p at 50NIp at 50NIGeotrichim candidum (3x)188.8.131.52253.121.56Monilinia sp. (2x)184.108.40.20656.253.12Mycosphaerella fijiensis (Sigatoka) (7 day, 3x)10.50.50.51.561.56Mycosphaerella fijiensis (8088/88) (7 day, 3x)0.120.250.060.50.1250.125Penicillium roqueforti (3x)0.510.51.561.561.56Pestalotiopsis microspora NE-32p at 50p at 50p at 50NININIPhoma chrysamthecola------Phyllosticta maydisp at 50p at 5050p at 50p at 50p at 50Phytophthora cactorumNINININININIPhytophthora cinnamomiNINININININIPhytophthora parasiticaNINININININIPhytophthora ultimumNINININININIRhizoctonia solani (2x)6.256.256.256.2525p at 50Sclerotinia sclerotiorum (2x)NINIp at 50NINI50Septoria passerinii (3x)0.1250.060.060.50.060.06Septoria tritici (3x)0.25 .0.250.060.50.1250.125Stagonospora nodorumNINININININITapesia acuformis (2x)50NININININITapesia yallundae (2x)2512.5256.25256.25Ustilago maydis (3x)0.510.51.560.250.25Verticillium dahliae (2x)p at 505025NI25p at 50
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-PO4A-FBBB'CC'Alternaria helianthi (2x)25255025NINIAphanomyces sp. (2x)p at 50NI50NI50p at 50Bipolaris sorokinianaNINININININIBotrytis alli (2x)p at 50NININININICochliobolus carbonumNINININININIDiplodia natalensisNINININININIDrechslera portulacae (3x, 9 day)0.060.250.060.250.060.5Drechslera teresNINININININIDrechslera tritici-repentisp at 50NIp at 50p at 50NINIFusarium avenaceumNINININININIFusarium culmorumNINININININIFusarium oxysporum cubenseNINININININIFusarium solaniNINININININIGeotrichim candidum (3x)6.256.253.1225256.25Monilinia sp. (2x)12.512.56.252512.56.25Mycosphaerella fijiensis (Sigaioka) (3x, 21 days)11111.561.56Mycosphaerella fijiensis (8088/88) (3x, 21 days)0.250.250.1210.250.25Penicillium roqueforti (3x)220.127.116.11.5612.5p at 50Pestalotiopsis microspora NE-32NINIp at 50NININIPhoma chrysamthecolaNIp at 50p at 50p at 50NINIPhyllosticta maydisNINI50NININIPhytophthora parasiticaNINININININIPhytophthora cinnamomiNINININININIPhytophthora ultimumNINININININIPhytophthora cactorumNINININININIRhizoctonia solani (2x)12.550506.25p at 50p at 50Sclerotinia sclerotiorum (2x)NININININIp at 50Septoria tritici (3x)0.250.250.060.50.250.25Septoria passerinii (3x)0.120.060.060.50.060.06Stagonospora nodorumNINININININITapesia acuformis (2x)50NININININITapesia yallundae (2x)5025NI50NINIUstilago maydis (3x)18.104.22.168.25Verticillium dahliae (2x)p at 50p at 50p at 50NIp at 50p at 50
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, 1.56µg/ml. These
fungi 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 (PO4), B and C (9days). Two different isolates of Mycosphaerella fijiensis
responded differently to the lower doses of pseudomycins. The Sigatoka isolate appeared
to be inhibited by each of the pseudomycins down to ∼ 1 µg/ml. However, 8088/88
isolate was best inhibited by pseudomycin B, with no growth at 0.125 µg/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
µg/ml pseudomycin B, and S. passerinii showing no growth at 5 days at 0.0625
µg/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 µg/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 µg/ml at 5 days), Monilinia sp., best inhibited by B and C' (no growth
at 6.25 µg/ml at 5 days), Geotrichim candidum, best inhibited by B (no growth
at 3.12 µg/ml at 5 days) andPenicillium roqueforti, best inhibited by B'
(no growth at 1.56µg/ml at 5 days).
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.
Penicillum 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
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.
2. C. Potera. Science, 1994, 265, 605.
3. L. Harrision, D. B. Teplow, M. Rinaldi, G. A. Strobel. J. General Microbiology1991,
4. B. Lam, G. Strobel, L. Harrison, S. Lam. Proc. Natl. Acad Sci.
1987, 84, 6447-6451.
5. R. Scheffer, D. Elgersma, G. Strobel. Neth. J. Pl. Path.
1989, 95, 293-304.
6. R. Scheffer, D. Elgersma, L. A. DeWeger, G. Strobel. Neth. J. Pl. Path.
1989, 95, 281-292.
Verfahren zur Prävention oder zum Behandeln einer Infektion von Pflanzen und
Getreiden durch einen oder mehrere ascomycetösen Pilz(e) der GattungMycoshaerella
sp. oder seiner Imperfektstadien Septoria sp. oder Cercospora sp.,
welches das Verabreichen einer wirksamen Menge von einer oder mehreren Pseudomycin-Zusammensetzung(en)
an diese Pflanzen oder Getreide umfaßt.
Verfahren nach Anspruch 1, wobei der/die Pilz(e) Mycoshaerella frjiensis
Verfahren nach Anspruch 1 oder Anspruch 2, wobei das Pseudomycin aus
dem mit Pflanzen assoziierten Bakterium Pseudomonas syringae isoliert ist.
Verfahren nach Anspruch 3, wobei das Pseudomycin aus der Gruppe, bestehend aus
Pseudomycin A, Pseudomycin A', Pseudomycin B, Pseudomycin B', Pseudomycin C
und Pseudomycin C', ausgewählt ist
Verfahren nach einem der Ansprüche 1 bis 4, wobei das Pseudomycin auf die Pflanze
oder das Getreide als eine wäßrige Suspension, Lösung oder Emulsion mit einer
Konzentration im Bereich von etwa 1 bis 100 Mikrogramm pro ml aufgetragen wird.
Verfahren nach einem der Ansprüche 1 bis 5, wobei die Pflanzen und Getreide
aus der Gruppe, bestehend aus Bananen, Kochbananen, Sonnenblume, Zuckerrüben, Gerste,
Zwiebel, Mais, Trauben, Portulak, Weizen, Tomate und Mais, ausgewählt sind.
Verwendung von einer oder mehreren Pseudomycin-Zusammensetzung(en) für
die Prävention oder die Behandlung der Black Sigatoka-Erkrankung bei Pflanzen und
Verwendung nach Anspruch 7, wobei die Pseudomycin-Zusammensetzung Pseudomycin,
das von dem mit Pflanzen assoziierten Bakterium Pseudomonas syringae isoliert
Verwendung nach Anspruch 8, wobei das Pseudomycin aus der Gruppe, bestehend
aus Pseudomycin A, Pseudomycin A', Pseudomycin B, Pseudomycin B', Pseudomycin
C und Pseudomycin C', ausgewählt ist.
Verwendung nach einem der Ansprüche 7 bis 9, wobei die Pseudomycin-Zusammensetzung
auf die Pflanze oder das Getreide als eine wäßrige Suspension, Lösung oder
Emulsion mit einer Konzentration des Pseudomycins in einem Bereich von etwa 1 bis
100 Mikrogramm pro ml aufgetragen wird.
A method for the prevention or treatment of plants and crops of infection by
one or more Ascomycetous fungi of the genus Mycoshaerella sp, or its imperfect
stages Septoria sp. or Cercospora sp., which comprise applying to
said plants or crops an effective amount of one or more Pseudomycin compositions.
The method according to claim 1, wherein the fungi is Mycoshaerella fijiensis.
The method according to claim 1, or claim 2, wherein the Pseudomycin
is isolated from the plant associated bacterium Pseudomonas syringae.
The method according to claim 3, wherein said the pseudomycin is selected from
the group consists of Pseudomycin A, Pseudomycin A', Pseudomycin B, Pseudomycin
B', Pseudomycin C, Pseudomycin C'.
The method according any one of claims 1 to 4, wherein the pseudomycin is applied
to said plant or crop as an aqueous suspension, solution or emulsion with a concentration
ranging from about 1 to 100 micrograms per ml.
The method according any one of claims 1 to 5, wherein said plants and crops
are selected from the group consisting of bananas, plantains, sunflower, sugar beets,
barley, onion, corn, grapes, portulaca, wheat, tomato and com.
Use of one or more Pseudomycin compositions for the prevention or treatment
of Black Sigatoka Disease in plants and crops.
The use according to claim 7, wherein the Pseudomycin composition comprises
Pseudomycin isolated from the plant associated bacterium, Pseudomonas syringae.
The use according to claim 8, wherein the Pseudomycin is selected from the group
consisting of Pseudomycin A, Pseudomycin A', Pseudomycin B, Pseudomycin B', Pseudomycin
C, Pseudomycin C'.
The use according to any one of claims 7 to 9, wherein the Pseudomycin composition
is applied to said plant or crop as an aqueous suspension, solution or emulsion
having a concentration of Pseudomycin ranging from about 1 to 100 micrograms per
Procédé pour la prévention ou le traitement des plantes et des cultures de l'infection
par un ou plusieurs des champignons Ascomycètes du genre Mycoshaerella sp,
ou de ses stades imparfaits Septoria sp. ou Cercospora sp., qui comprend
l'application auxdites plantes ou cultures d'une quantité efficace d'une ou plusieurs
compositions de Pseudomycine.
Procédé selon la revendication 1, dans lequel le champignon estMycoshaerella
Procédé selon la revendication 1, ou la revendication 2, dans lequel on isole
la Pseudomycine de la bactérie associée à la plante Pseudomonas syringae.
Procédé selon la revendication 3, dans lequel on choisit la pseudomycine dans
le groupe constitué de la Pseudomycine A, Pseudomycine A', Pseudomycine B, Pseudomycine
B', Pseudomycine C, Pseudomycine C'.
Procédé selon l'une quelconque des revendications 1 à 4, dans lequel on applique
la pseudomycine à ladite plante ou culture sous la forme d'une suspension aqueuse,
d'une solution ou d'une émulsion ayant une concentration dans une plage d'environ
1 à 100 microgrammes par ml.
Procédé selon l'une quelconque des revendications 1 à 5, dans lequel on choisit
lesdites plantes et cultures dans le groupe constitué des bananes, plantains, tournesol,
betteraves, orge, oignon, maïs, raisins, portulaca, blé, tomate et maïs.
Utilisation d'une ou de plusieurs compositions de Pseudomycine pour la
prévention ou le traitement de la maladie Black Sigatoka dans les plantes et les
Utilisation selon la revendication 7, dans laquelle la composition dePseudomycine
comprend de la Pseudomycine isolée de la bactérie associée à la plante,Pseudomonas
Utilisation selon la revendication 8, dans laquelle on choisit la Pseudomycine
dans le groupe constitué de la Pseudomycine A, Pseudomycine A', Pseudomycine
B, Pseudomycine B', Pseudomycine C, Pseudomycine C'.
Utilisation selon l'une quelconque des revendications 7 à 9, dans laquelle on
applique la composition de Pseudomycine à ladite plante ou culture sous la forme
d'une suspension aqueuse, d'une solution ou d'une émulsion avant une concentration
de Pseudomycine dans une plage d'environ 1 à 100 microgrammes par ml.