U.S. patent application number 14/077935 was filed with the patent office on 2014-05-08 for pesticidal compositions comprising 4,5-dihydroxyindan-1-one.
This patent application is currently assigned to YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREBREW UNINVERSITY OF JERUSALM LT. The applicant listed for this patent is Izhak Bilkis, Uri Gerson, Zohar Kerem, Zahi Paz, Abraham Sztejnberg. Invention is credited to Izhak Bilkis, Uri Gerson, Zohar Kerem, Zahi Paz, Abraham Sztejnberg.
Application Number | 20140128257 14/077935 |
Document ID | / |
Family ID | 43759123 |
Filed Date | 2014-05-08 |
United States Patent
Application |
20140128257 |
Kind Code |
A1 |
Sztejnberg; Abraham ; et
al. |
May 8, 2014 |
PESTICIDAL COMPOSITIONS COMPRISING 4,5-DIHYDROXYINDAN-1-ONE
Abstract
Provided are pesticidal compositions comprising
4,5-dihydroxyindan-1-one or derivatives thereof for protecting
important crops against mites, fungi, and bacteria. The
compositions may be manufactured by fractionating fungal
extracts.
Inventors: |
Sztejnberg; Abraham;
(Rehovot, IL) ; Gerson; Uri; (Rehovot, IL)
; Paz; Zahi; (Gedera, IL) ; Kerem; Zohar;
(Rehovot, IL) ; Bilkis; Izhak; (Gedera,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sztejnberg; Abraham
Gerson; Uri
Paz; Zahi
Kerem; Zohar
Bilkis; Izhak |
Rehovot
Rehovot
Gedera
Rehovot
Gedera |
|
IL
IL
IL
IL
IL |
|
|
Assignee: |
YISSUM RESEARCH DEVELOPMENT COMPANY
OF THE HEBREBREW UNINVERSITY OF JERUSALM LT
Jerusalem
IL
|
Family ID: |
43759123 |
Appl. No.: |
14/077935 |
Filed: |
November 12, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13496469 |
Mar 15, 2012 |
8592344 |
|
|
14077935 |
|
|
|
|
Current U.S.
Class: |
504/101 ;
424/195.16; 504/116.1; 514/681 |
Current CPC
Class: |
A01N 63/30 20200101;
A01N 63/30 20200101; A01N 35/06 20130101; A01N 35/06 20130101; A01N
2300/00 20130101; A01N 63/30 20200101; A01N 2300/00 20130101; A01N
2300/00 20130101 |
Class at
Publication: |
504/101 ;
514/681; 424/195.16; 504/116.1 |
International
Class: |
A01N 35/06 20060101
A01N035/06; A01N 63/04 20060101 A01N063/04 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 13, 2010 |
IL |
PCT/IL2010/000747 |
Claims
1. A method of controlling fungal or bacterial infestation in a
plant susceptible thereto, comprising applying onto the plant or in
the vicinity of said plant infested with fungi or bacteria a
composition comprising a compound of formula I ##STR00005## wherein
R.sub.1 and R.sub.2 are independently selected from H, C.sub.1-18
alkyl, and C.sub.1-18 acyl.
2. A method according to claim 1, comprising a compound of formula
I ##STR00006## wherein R.sub.1 and R.sub.2 are independently
selected from H, C.sub.1-18 alkyl, and C.sub.1-18 acyl, and at
least one component selected from the group consisting of
agriculturally acceptable carrier, diluent, emulsifier, dispersant,
and an additional active ingredient selected from herbicides,
insecticides, growth stimulators, and fertilizers.
3. A method according to claim 1, wherein said plant comprises
fruit, vegetable, or ornamental flower.
4. A method of controlling bacterial infestation according to claim
1, comprising applying onto the plant or in the vicinity of said
plant a composition comprising an extract of a fungus selected from
Meira argovae, Meira geulakonigae, and Acaromyces ingoldii, or
biologically active products derived from the extract
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of and claims priority to
U.S. application Ser. No. 13/496,469, filed on Mar. 15, 2012, which
claims priority to PCT Application No. PCT/IL00747, filed on Sep.
13, 2010, which claims priority to US Application No. 61/242,829,
filed Sep. 16, 2009, the contents of each of which are incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to pesticidal compositions
containing a fungal metabolite based on dihydroxyindane,
particularly compositions for controlling mites, fungi, and
bacteria causing damage to important crops.
BACKGROUND OF THE INVENTION
[0003] Plant diseases caused by mites, bacteria and fungi have a
significant adverse impact on the production of important crops
worldwide. The most important mites include spider mites, causing
damages to many fruits, vegetables, and flowers. Examples include
the two-spotted spider mite and the citrus rust mite. Chemical
control has met with increasing difficulties, among others the
development of resistance to pesticides, and to regulatory issues.
Fungi secrete a wide range of secondary metabolites, of which many
are toxic to other organisms and microorganisms, and may be used
for biocontrol in agricultural systems. It is therefore an object
of this invention to provide a method of controlling crop pests,
employing fungal metabolites.
[0004] It is another object of this invention to provide a
pesticide for protecting important crops against mites, bacteria
and fungi.
[0005] It is still another object of this invention to provide an
acaricide derived from fungi.
[0006] It is a further object of this invention to provide an
acaricidal composition for protecting plants, including their
fruits, susceptible to mites, bacteria and fungi.
[0007] It is a still further object of this invention to provide a
method of controlling and preventing the infestation by mites and
eventually other pests (including bacteria and fungi), comprising
applying fungus-derived components.
[0008] It is also an object of this invention to provide a method
of preparing a pesticidal formulation for protecting plants
susceptible to mites, bacteria and fungi, such as citrus
fruits.
[0009] Other objects and advantages of present invention will
appear as description proceeds.
SUMMARY OF THE INVENTION
[0010] The present invention provides a pesticidal composition
comprising as an active ingredient a compound of formula I
##STR00001##
wherein R.sub.1 and R.sub.2 are independently selected from H,
C.sub.1-18 alkyl, and C.sub.1-18 acyl. As the present invention
demonstrates the pesticidal activity of 4,5-dihydroxyindan-1-one, a
skilled person will appreciate that derivatives which are easily
metabolized in the pests' bodies to 4,5-dihydroxyindan-1-one will
also be toxic for said pests. An example of such derivatives is
4,5-dihydroxyindan-1-one esterified on one or both hydroxyls by a
carboxylic acid. In a preferred embodiment, said pesticidal
composition comprises an extract from a fungus. A preferred fungus
is Meira argovae. A pesticidal composition according to the
invention preferably exhibits acaricides and fungicidal activity.
Said pesticidal composition further preferably exhibits bactericide
activity, such as against Agrobacterium tumefaciens.
[0011] The invention is directed to a composition for the use in
controlling or preventing mite, fungi or bacterial infestation in a
commercial plant, such as important crops, for example comprising
fruit trees, vegetables, ornamental flowers, etc.
[0012] The mites may be, for example, spider mites. The composition
may further comprise at least one component selected from the group
consisting of agriculturally acceptable carrier, diluent,
emulsifier, dispersant, and an additional active ingredient
selected from herbicides, insecticides, growth stimulators, and
fertilizers.
[0013] In another aspect of the invention, the present invention
concerns an antibacterial composition comprising an extract from at
least one of the fungus Meira argovae, Meira geulakonigae, or
Acaromyces ingoldii progeny, mutants or variants thereof retaining
the activity against bacteria, or biological products derived from
said extracts.
[0014] The present invention further concerns a method for
controlling bacterial infestation in plants comprising applying to
the plant or to the vicinity of the plant an antibacterial
composition comprising an extract from at least one of the fungi
Meira argovae, Meira geulakonigae, or Acaromyces ingoldii progeny,
mutants or variants thereof retaining the activity against
bacteria, or biological products derived from said extracts.
[0015] Preferably the extract is from the fungus Meira argovae.
[0016] The fungus Meira argovae, is preferably the fungus
designated CBS Accession No. 110053, progeny, mutants or variants
thereof that retain the antibacterial activity.
[0017] The Meira geulakonigae is preferably designated CBS
Accession No. 110052, progeny, mutants or variants thereof
retaining the anticaterial activity.
[0018] The Acaromyces ingoldii, is preferably the fungus designated
CBS Accession No. 110050, progeny, mutants or variants thereof
retaining the antibacterial activity.
[0019] Preferably the composition comprises an extract from the
fungus Meira argovae, and it exhibits in addition to antibacterial
activity also acaricidal and fungicidal activities. In an important
embodiment, the composition according to the invention comprises a
compound of formula II
##STR00002##
[0020] The invention provides a method of controlling mite, fungi
or bacterial infestation in a plant susceptible thereto, comprising
applying onto the plant or in the vicinity of said plant a
composition comprising a compound of formula I, as described above,
for example a compound of formula II. In the method according to
the invention, said composition has preferably acaricidal,
fungicidal and bactericidal activities; said plant usually
comprises fruits, vegetables, or ornamental flowers. Examples of
the mites to be controlled may include, without any limitations,
the two-spotted spider mite and the citrus rust mite.
[0021] The invention relates to a method of manufacturing the
composition comprising a compound of formula I
##STR00003##
wherein R.sub.1 and R.sub.2 are independently selected from H,
C.sub.1-18 alkyl, and C.sub.1-18 acyl, comprising cultivating a
fungus, extracting the culture medium, and chromatographically
separating the metabolites of said fungus released into said
medium, and further optionally derivatizing said metabolite. In a
preferred embodiment of the invention, the method of manufacturing
the pesticidal composition comprises producing a metabolite of
formula II
##STR00004##
wherein the production is increased by adjusting the pH in the
cultivating medium.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The above and other characteristics and advantages of the
invention will be more readily apparent through the following
examples, and with reference to the appended drawings, wherein:
[0023] FIG. 1. shows separation of an active fungal agent; FIG. 1A
is HPLC chromatogram of extracted metabolites from Meira argovae;
FIG. 1B is a UV Spectrum related to the second major peak, of which
elution starts at 8.46 min; FIG. 1C shows applying the material
eluted in said second major peak as the bioactive toxin inhibiting
the growth of Agrobacterium tumefaciens;
[0024] FIG. 2. shows the proposed structure of the active agent,
called argovin herein, namely 4,5-dihydroxyindan-1-one (molecular
formula, C.sub.9H.sub.7O.sub.3), being a mite-antagonistic compound
secreted by Meira argovae;
[0025] FIG. 3. is a graph showing colony areas of Meira argovae
colonies grown on phosphate buffered media, with initial pH values
of 5, 6, or 7; different letters denote significant differences
between the treatments (at p<0.05);
[0026] FIG. 4. is a graph showing the pH changes induced by Meira
argovae colony in non-buffered media; initial pH of media was 4.0
(.diamond-solid.), 4.5 (.box-solid.), 5.0 (.tangle-solidup.) or 6.0
( ), respectively; the pH changes were monitored by readings at
equal distances from the colonies edges;
[0027] FIG. 5. is a graph showing pH-dependent toxin production by
Meira argovae, as detected by HPLC; the fungus was grown in liquid
phosphate-buffered media, with initial pH values of 5, 6, or 7, or
control (Con), which is a non-buffered medium (initial pH of 5.0);
different letters denote significant differences between treatment
(at p<0.05);
[0028] FIG. 6. shows the dose response of the citrus rust mite to
RPLC-purified toxin (argovin) of Meira argovae;
[0029] FIG. 7. is a graph showing the citrus rust mite mortality in
response to applications of Meira argovae conidia (10.sup.8,
ml.sup.-1) on ripe grapefruits (.diamond-solid.), untreated ripe
grapefruits (control) (.tangle-solidup.), and fungal-treated raw
fruits (.box-solid.), compared to raw untreated fruits (control) (
); the pH values of grapefruit peels were measured in both ripe and
raw fruits;
[0030] FIG. 8. shows the effect of extracts, prepared from the
dishes on which Acaromyces ingoldii (Ai), Meira argovae (Ma) and
Meira geulakonigii (Mg) had developed, on the colony growth of
seven phytopathogenic fungi: 1. Rhizoctonia solani; 2. Sclerotium
rolfsii; 3. Sclerotinia sclerotiorum; 4. Colletotrichum
gloeosporioides; 5. Pencillium digitatum; 6. Fusarium mangiferae
and 7. Phytophthora citrophthora. All treatments were significantly
different from the controls at P<0.05, except for the effect of
M. geulakonigii on F. mangiferae (6);
[0031] FIG. 9. shows the effect of extracts, prepared from the
dishes on which Acaromyces ingoldii (Ai), Meira argovae (Ma) and
Meira geulakonigii (Mg) had developed (the symbols are the same as
in FIG. 8), on the development of mycelia from the sclerotia of
Sclerotina sclerotiorum (FIG. 9A) and Sclerotium rolfsii (FIG. 9B);
the vertical lines indicate SE;
[0032] FIG. 10. presents the extent of tomato leaflet coverage by
Sclerotina sclerotiorum as affected by a spray of
1-5.times.10.sup.8 ml.sup.-1 suspensions of the blastoconidia of
Acaromyces ingoldii (Ai), Meira argovae (Ma), and Meira
geulakonigii (Mg) in deionized water; control leaves were treated
only with deionized water; the extent of coverage of the control
leaflets differed significantly from the effect of all FBAs; the
vertical lines indicate SE and the asterisks indicate significant
differences from the controls at P<0.05;
[0033] FIG. 11. presents the extent of green mold infection caused
by Pencillium digitatum to oranges pre-treated by a spray of
1-5.times.10.sup.8 ml.sup.-1 suspensions of the blastoconidia of
Acaromyces ingoldii (Ai), Meira argovae (Ma) and Meira geulakonigii
(Mg) in deionized water; control oranges were treated only with
deionized water; the extent of infection of the control oranges
differed significantly from that of the effect of all FBAs; the
vertical lines indicate SE;
[0034] FIG. 12. presents SEM micrographs of the hyphae of the FBAs
penetrating into the skin of an orange (Citrus sinensis cv. New
Hall); FIG. 12A shows Acaromyces ingoldii, FIG. 12B Meira argovae,
and FIG. 12C Meira geulakonigii; large pits in the middle of the
micrographs are stomata; arrows headed by an H point at the hyphae
of the FBAs; the bars in the top left corner represent 10 .mu.m;
and
[0035] FIG. 13. shows the inhibitory effect of the FBAs on
Pencillium digitatum (FIGS. 13A and 13C) and on Sclerotinia
sclerotiorum (FIGS. 13B and 13D), prior to heating in boiling water
(FIGS. 13A and 13B) and after heating (FIGS. 13C and 13D); all
treatments were significantly different from the controls at
P<0.05; the vertical lines shows SE.
DETAILED DESCRIPTION OF THE INVENTION
[0036] It has now been found that an HPLC fraction of the crude
extract from the fungus Meira argovae exhibits a surprisingly
strong acaricide activity. Furthermore, the same fraction shows
also an antibacterial activity, when checked on an important crop
pest, Agrobacterium tumefaciens. Such an unexpected advantageous
combination of bioactivities forms an excellent base for
manufacturing new pesticidal formulations.
[0037] Metabolites of the fungus Meira argovae were assayed as
antagonists of mites and bacteria. Separation of extracted fungal
metabolites by reversed phase liquid chromatography (RPLC), with
subsequent testing of the obtained fractions, allowed to the
present inventors to isolate a single mite-antagonistic fraction
that comprises one major component. This active compound, named
"argovin" herein, was identified by analysis of its spectral
characteristics as 4.5-dihydroxyindan-1-one. This compound has been
previously described only as a product or intermediate of chemical
reactions; here, the compound has been isolated as a naturally
occurring material, from a metabolite mixture. The growth rate of
the fungus was higher at a neutral pH than at an acidic one. Meira
argovae adjusts the pH of its media to values optimal for its
colony growth and toxic secretions. RPLC-cleaned argovin, at 0.2
mg/ml, killed 100% of a population of the citrus rust mite,
Phyllocoptruta oleivora. Conidia of M. argovae, when applied onto
mite-infested ripe grapefruits, caused high mite mortality, as
compared to mortality on infested unripe grapefruits. This may have
been due to differences in the pH values of the peels: 6.7 for the
ripe fruit and 5.6 for the unripe one. The inventors conclude that
the fungus exploits the changing pH conditions on grapefruits for
maximal toxin secretion, thereby causing higher mite mortality
rates. This trait may be used and manipulated to control citrus
rust mites in the field, as well as for toxin production for
industrial and pharmaceutical uses. Agrovin is, thus, a novel
natural product, antagonistic to mites, produced by the fungus
Meira argovae, whose secretion is affected by ambient pH.
Surprisingly and advantageously, the same material turned out to
have also a bactericide activity, when checked on Agrobacterium
tumefaciens--the bacterial causal agent of tumor formation in many
agriculturally important plants, belonging among economically most
important crop pathogens.
[0038] The invention thus relates to a pesticidal formulation
comprising a component derived from a fungal extract. Particularly,
Meira argovae fungus was employed; the crude fungal extract was
then chromatographed and bio-assayed. Three major peaks, detected
by the RPLC analysis (FIG. 1A), were collected and used for
bioassays, along with other non-UV detected fractions that had
eluted from the RPLC. Only one peak, eluted at 8.46 min, whose UV
spectrum was characterized by .lamda.max=205, 235 and 285 nm (FIG.
1B), was active against A. tumefaciens (FIG. 1C). The fraction
characterized by .lamda.max=205, 235 and 285 nm was collected,
lyophilized and subjected to analysis by MS and MS/MS and by
different modifications of the NMR spectroscopy. M-H mass of the
compound was 163.0401 (molecular formula, C9H7O3). According to the
MS/MS spectrum, the major losses of the M-H peak are H2, H2O, CO
and CH2=C.dbd.O. The 1H-NMR spectrum of the compound in CD3OD
solution shows four groups of protons: multiplet at .delta.=2.66
(2H), multiplet at .delta.=3.04 (2H), doublet at .delta.=6.86 (1H,
J=8 Hz) and doublet at .delta.=7.17 (1H, J=8 Hz). The 13C-NMR
spectrum shows 9 different signals at .delta.: 23.18 (CH2 according
to DEPT spectra), 37.45 (CH2 according to DEPT spectra), 116.76 (CH
according to DEPT spectra), 117.09 (CH according to DEPT spectra),
130.69, 142.92, 144.58, 152.89, 208.99. The HQMC spectrum allowed
us to assign the protons with .delta.=2.66 to carbon atom with
.delta.=37.45, protons with .delta.=3.04 to carbon atom with
.delta.=23.18, proton with .delta.=6.86 to carbon atom with
.delta.=116.76 proton with .delta.=7.17 to carbon atom with
.delta.=117.09. Evidently, the carbon with .delta.=208.99 belongs
to a carbonyl group. According to the COSY spectra, the two CH2
groups are linked to each other, and the same holds for the two
CH-groups. All the above presented data lead us to the suggestion
that the analyzed compound has an indan-1-one skeleton with two
hydroxyl groups linked to aromatic moiety. The HMBC spectrum i)
H(2.66) was found to be in interaction with C(23.18, strong),
C(130.69, weak), C(144.58, strong), C(208.99, strong); ii) H(3.04)
was in interaction with C(37.45, strong), C(130.69, strong),
C(142.92, strong), C(144.58, strong), C(152.89, strong), C(208.99,
strong); iii) H(6.86) was found to be in interaction with C(130.69,
strong), C(142.92, strong), C(152.89, weak); iv) H(7.17) was found
to be in interaction with C(144.58, strong), C(152.89, weak),
C(208.99, strong). With the following assignment of the NMR
signals: 23.18 (23.54)-C(2), 37.45(37.20)-(C3),
116.76(115.07)-(C6), 117.09(119.01)-(C7), 130.69(128.89)-(C7a),
142.92(142.23)-(C4), 144.58(142.93)-(C3a), 152.89(152.29)-(C5),
208.99-(C1). The assignment was supported by good agreement between
the measured and calculated .sup.13C chemical shifts (shown in the
parentheses), the calculations were performed using the ACD
(version 10.0) .sup.13C-Predictor during the visit of second author
in the laboratory of Prof. G. Gescheidt, Graz (University of
Technology, Graz, Austria). The additional data suggest that the
analyzed compound structure is 4,5-dihydroxyindan-1-one (syn.
4,5-dihydroxy-1-indanone; 4,5-dihydroxy-2,3-dihydro-1H-inden-1-one)
and was consequently named argovin (FIG. 2). The major metabolite
of the toxic fraction was thus identified.
[0039] The effect of different pH values on fungal growth was
examined. Fastest fungal growth took place on media buffered to pH
6.0 and 7.0 (attaining an average of 311 mm.sup.2 and 327 mm.sup.2
in diameter, respectively), as compared to 222 mm.sup.2 at pH 5.0
(FIG. 3). Measuring changes in the pH values (InLab.RTM. 427
microelectrode) in the growth media indicated higher values near
the colonies' edges, as compared to other sites. The initial pH of
the media, which came to 4.0, rose to 6.0 after 21 days (measured
at 20 mm from the colonies' edge). When the initial pH values were
4.5, 5.0 or 6.0, they ascended to 7.2, 7.3 and 7.3, respectively
(FIG. 4). Further, the effect of pH values on the quantity of
argovin secretions was characterized. The highest amount of toxin
secretion was measured in colonies grown at pH 7.0 (5.2 mg/ml)
buffered media, as compared to that obtained when the fungus was
grown at pH 6.0 (3.1 mg/ml) or at pH 5.0 (0.98 mg/ml). The amount
of secretions in the non-buffered control was not statistically
different (1.99 mg/ml) from that obtained at pH 6.0. The pH in the
non-buffered controls rose during the experiment from 5.0 to 6.0
(FIG. 5).
[0040] The effect of the changing pH of grapefruit peel on the
antagonism of M. argovae to the citrus rust mite was checked. The
pH value of non-ripe grapefruit peels was close to 5.5, and that of
ripe fruits, 6.5. Following the application of fungal conidia,
highest mite mortality (85%) was seen on ripe fruits, as compared
to 78% mortality (significantly different at 0.05) on non-ripe
fruits. In the control treatment (water only), which included CRM
on ripe or non-ripe fruits, mortality came only to 11 and 9%,
respectively. These data suggest an association between the peel's
pH level and mite mortality (R2=0.81) (FIG. 7).
[0041] The dose response of the citrus rust mite to argovin
concentrations was measured. The active fraction (at 0.01 mg/ml)
caused 39% mortality (vs. 6% in the control), rising to 100%
mortality at 0.2 mg/ml (FIG. 6).
[0042] The present invention thus provides the means to fight the
pests, such as A. tumefaciens and the citrus rust mite (CRM), by
employing a compound secreted by Meira argovae. An RPLC method is
provided for detecting and isolating the bioactive fraction in the
fungal secretions, particularly wherein the fungus comprises M.
argovae. The provided bioassay is very practical because the
utilization of A. tumefaciens allows the overnight reading of the
results. The identification and characterization of the fungal
substance 4,5-dihydroxyindan-1-one (termed here argovin) was
enabled, the substance being toxic to A. tumefaciens and to the
CRM.
[0043] No report has been found by the inventors about secreting
the compound argovin by any organism, nor about its biological
activity. Singh et al. (1989) described this compound as an
intermediate between 4,5-dimethoxyindan-1-one and
4,5-dibenzyloxy-2,3-dihydro-1-H-indaen-1-one, in a study aimed at
understanding the stereochemical assignments of these compounds,
without relating to biological activities. A similar compound,
5,6-dihydroxyindan-1-one, was reported as an intermediate in the
bromination of 5,6-dimetoxyindan-1-one bromination, without
relating to any biological activity (Choi and Ma, 2007). Different
derivatives of 1-indanone were reported to have an adverse effect
on breast cancer growth in human cells in vitro (Somers-Edgar and
Rosengren, 2009). According to another study, derivatives of
1-indanone have pharmaceutical uses against Alzheimer's disease (da
Silva et al., 2006).
[0044] Many fungi that served as biocontrol agents (or potential
biocontrol agents) secrete toxic secondary metabolites that are the
main, or the only, mode of antagonistic activity (Vey et al.,
2001). For example, the yeast-like fungus Pseudozyma flocculosa, a
species related to Meira argovae, is a potential biocontrol agent
that secrets toxic fatty acids (Avis and Belanger, 2001) and a
cellobiose lipid (Cheng et al., 2003), which were verified as the
main modes of action against cucumber powdery mildew and Phomopsis
sp. (Avis and Belanger, 2001; Cheng et al., 2003).
[0045] Changes in pH values also affect growth rates in other
fungal systems. For instance, Coniothyrium minitans, a mycoparasite
of Sclerotinia spp., grows better and produces more toxins under
acidic than neutral-alkali conditions (Yang et al., 2008). The
entomopathogenic fungus Metarhizium anisopliae regulates the
ambient pH, which activates its cuticle-degrading enzymes and
amplifies a toxic secretion following penetration (St Leger et al.,
1999). In the instant case, it seems that the changes in the pH of
the grapefruit peel during ripening contributes to fungal
development and allows argonin secretion to occur at a higher rate.
In this work, it is demonstrated that M. argovae changes the pH of
its environment to neutral when grown on artificial media, and as a
result secretes higher amounts of argovin, as compared to more
acidic conditions. Argovin directly causes mite death, as shown by
its enhanced mortality upon increasing the amounts of toxin
applied. The fact that argovin is capable of inhibiting A.
tumefaciens as well as mites (e.g. prokaryote and eukaryote
organisms) suggests that it may have pharmaceutical along with
agricultural uses. Little information is available on fungal
mycotoxins, especially those of the Ustilaginomycetidae (Vey et
al., 2001), to which M. argovae belongs. This fungus is shown as
having a great potential of becoming a biocontrol agent against
herbivorous mites and other plant pathogens, including fungi and
bacteria. The link between grapefruit peel pH and the antagonistic
activity of the fungus can be manipulated when using it against
citrus mites. Applying the fungus in an alkali medium/formulation
may lead to higher pest mortality, regardless of the environmental
pH. This biological trait may also be exploited in order to obtain
more argovin when producing it for industrial and pharmaceutical
purposes. This work identifies and characterizes the toxic
secretion of M. argovae and its dependence on the ambient pH. For
the first time, argovin is described as a living organism
metabolite (fungal), beside demonstrating its effects in reducing
pest-mite populations.
[0046] In a preferred embodiment of the present invention, the
formulation is used in protecting the crops against mites, fungi
and/or bacteria, the formulation comprising a fungal extract or a
component isolated from such extract, particularly an extract from
M. argovae. In one embodiment, said isolated component is
derivatized by known chemical processes. Preferably, a fungal
extract or its component is applied onto plants to be protected or
in their vicinity, the extract comprising 4,5-dihydroxyindan-1-one
or a derivative thereof. Said derivative may comprise an ester or
ether. Particularly, derivatives that are easily hydrolyzed in the
pest bodies to provide free 4,5-dihydroxyindan-1-one are
preferred.
[0047] Provided is a pesticidal formulation comprising
4,5-dihydroxyindan-1-one and agriculturally acceptable carriers,
auxiliaries or diluents, such as solvents, emulsifiers and
dispersants or surfactants. In some embodiments, the formulation of
the invention may comprise another pesticide providing a
synergistic effect against mites, fungi and/or bacteria, or
providing activity against additional pests. For some uses, the
formulation may further contain active ingredients, such as
herbicides, insecticides, growth stimulators, and fertilizers.
[0048] In yet another aspect of the invention, a method for
controlling and/or preventing infestation by mites, fungi and
bacteria in agriculturally important plants is provided. In one
embodiment of the invention, a method for controlling and/or
preventing infestation by mites, fungi and/or bacteria in
agriculturally important plants is provided.
[0049] For the purpose of carrying out the invention, the
acaricidal, fungicidal and bactericidal suspension or solution can
be applied by any way known in the art of pesticide use and
agricultural protection.
[0050] The invention will be further described and illustrated in
the following examples.
EXAMPLES
Fungal and Bacterial Strains, Growth Conditions and Crude Fungal
Metabolite Extraction
[0051] Meira argovae (strain AS005, Boekhout et al., 2003) was
employed. The fungus was routinely grown on potato dextrose agar
(PDA, Difco, Detroit) at 25.degree. C., and maintained at 4.degree.
C. until used. For metabolite extraction, the fungus was grown in
30 ml yeast malt peptone dextrose (YMPD) broth (Cheng et al., 2003)
for 28 days in the dark, with constant agitation (150 rpm).
Extraction of metabolites followed Duffy and Defago (1999), with
minor changes as described previously (Paz et al., 2007b). A
culture of Agrobacterium tumefaciens (strain ID1) used for the
bioassays, was kindly provided by Prof. Leonid Chemin of the Hebrew
University of Jerusalem.
Source of the Citrus Rust Mites
[0052] The CRM population was obtained from Mineola Tangelo (C.
paradisi.times.C. reticulata) fruits at the Tzrifin farm, on the
coastal plain of Israel. No acaricides are being used at this farm,
making the location a suitable place for our purposes. In the
laboratory the pest was reared on young seedlings (6-8 weeks old)
of sour orange (Citrus aurantium).
Crude Extract Chromatography
[0053] Reversed phase liquid chromatography (RPLC) separation was
performed to detect biologically active fractions from the crude
fungal extract. The HPLC system (Thermo Separation Products,
Riviera Beach, Fla.) consisted of an auto-sampler (AS3000),
injector (1000, column oven (35.degree. C.), pump (P3000) and diode
array detector (UV6000). A reverse phase C18 column (250.times.4.6
mm, "Luna" 5.mu. Phenomenex, Torrance, Calif.) was employed.
Elution was performed using a linear gradient consisting of double
distilled water (ddH2O) and acetonitrile, initially an isocratic
step was employed for 3 min at 85% water, followed by a moderate
increase in acetonitrile to reach 17% in 14 min, at a flow rate of
1 ml/min. The crude extract was monitored at .lamda.max=210, 232
and 285 nm. Each of the detected fractions was collected and
bio-assayed (see below).
Assay of Antibacterial Activity and Crude Extract Effect on the
Citrus Rust Mite
[0054] The crude extract was initially assayed in vitro against A.
tumefaciens, because preliminary results had indicated that this
bacterium is sensitive to the extract and therefore suitable for
our bioassays. The bacterium was grown overnight in glass tubes
containing a 5 ml Lennox Broth (LB, Difco). Fifty .mu.l
overnight-grown bacterial suspensions were added to 5 ml of LB
containing 0.6% agar, and placed into Petri dishes (55 mm
diameter). Each of the eluted chromatographed fractions was dried
using a rotor evaporator, dissolved in 10 .mu.l of methanol,
pipetted into the Petri dishes and incubated at 28.degree. C. Each
dish was checked for inhibition haloes 24 hours later; 10 .mu.l
methanol alone was used as a control. This procedure was repeated
four times to confirm of the toxicity of the fractions. The
RPLC-cleaned fraction that was antagonistic to A. tumefaciens was
concurrently assayed against the citrus rust mite (describe
below).
MS and NMR Analysis of the Toxic Fraction
[0055] MS analysis was performed on a Bruker Daltonik micrOTOF-Q
mass spectrometer equipped with an ESI ion source. The following MS
parameters were used for the analysis: capillary voltage 4000 V,
nebulizer pressure 0.6 Bar, dry gas flow 5 l/min, dry gas
temperature 180.degree. C., ISCID energy 1 eV/z, ion energy 3 eV/z,
collision energy 10-40 eV/z, mass range 100-2500 Da. Samples were
analyzed in positive and negative mode; better results were
obtained by the latter. A Bruker "Avance" DRX-500 NMR spectrometer,
operating at 500 MHz for 1H and at 125 MHz for 13C, was used for
the NMR experiments; chemical shifts are expressed in 6 (parts per
million) referring to the solvent peaks .delta..sub.H 3.34 and
.delta..sub.X 49.0 for CD3OD; coupling constants, J, are in
Hz.DEPT, 1H-1H COSY, 1H-13C HSQC, and HMBC NMR experiments were
carried out using the conventional pulse sequences as described in
the literature.
The Effect of Different pH Values on Fungal Growth
[0056] In order to assess whether colony growth is affected by
ambient pH, a six mm plaque from the edge of a fungal colony (on
PDA) was placed in the center of 90 mm Petri dishes containing
buffered YMPD with 2% agar (The buffer solution (citrate/phosphate)
contained 0.1 M citric acid and its pH was adjusted to the levels
of 5.0, 6.0 or 7.0 by adding amounts of 0.2 M Na2HPO4, as needed.).
These Petri dishes (5 replicates) were incubated for 21 days at
25.degree. C. in the dark. The diameter of each colony was measured
in order to obtain its growth area. Alizarin S red dye (Sigma)
(0.2%) was added onto each dish before colony measurement; this dye
is known to signal changes in acidity by forming a red halo when
the pH rises above 5.8. The actual pH values were measured by a pH
meter, with an InLab.RTM. 427 microelectrode at equal distances (5
mm) from the colonies edges. The fungus was also grown for 21 days
at 25.degree. C. in a non-buffered YMPD (2% agar) medium, whose pH
values were adjusted to 4.0, 4.5, 5.0 or 6.0. The pH values were
recorded at increasing distances from the colonies' edges (0.5,
1.0, 1.5 and 2.0 cm).
Effect of pH Values on the Quantity of Toxic Secretions
[0057] The purpose of this experiment was to determine the effect
of the pH of the media on toxin secretions and to establish the
optimal pH conditions for the secretion. A suspension of three ml
(1.times.10.sup.8 spores/ml) in ddH2O was added to 30 ml buffered
(phosphate/citrate, as above) YMPD in Erlenmeyer flasks. The flasks
were constantly agitated (150 rpm) for five days in the dark.
Non-buffered YMPD media served as controls. The amounts of the
extracted toxic metabolite resulting from each pH treatment were
measured with the RPLC system.
The Dose Response of the Citrus Rust Mite to Purified Toxin
Concentrations
[0058] In order to determine the CRM dose response to the toxin,
0.01, 0.02, 0.05, 0.1, 0.2 and 0.4 mg/ml of RPLC-cleaned toxin,
dissolved in a two ml volume of ddH2O, was sprayed on the CRM
(thirty CRMs/leaf, in four replicates). The infested seedlings were
incubated at 25.degree. C., (12L:12D). Mite mortality rates were
recorded 24 h later.
the Effect of the Changing pH of Grapefruit Peel on the Antagonism
of M. argovae to the Citrus Rest Mite
[0059] This experiment was conducted in order to explore the effect
of the pH of grapefruit peels on the fungal antagonism against the
CRM. Ten ripe and ten non-ripe red grapefruits (Citrus paradisi cv.
Star Ruby) were collected at Tzrifin farm, as above, and the mites
were from young seedlings, as noted. The pH of the fruits' outer
peel layer was measured, as above, with an InLab.RTM. 427
microelectrode (Mettler Toledo, Schwerzenbach, Switzerland). Each
grapefruit was infested by about 30 mites. Treatments included the
spray of a fungal suspension (1.times.108) on 5 ripe and 5 non-ripe
infested fruits and a water treated control, also on 5 ripe and 5
non-ripe fruits. CRM mortality was recorded five days
post-treatment (at 25.degree. C., 12L:12D; light: dark).
Statistical Analysis
[0060] Each experiment was conducted twice with 3-5 replicates.
Data obtained were analyzed by ANOVA Tukey's test (P=0.05) using
JMP 8.0 Software (SAS Institute, Cary, N.C.,). For the
mite-infested red grapefruits experiment, a regression analysis was
conducted.
The Effect of Three Basidiomycotine Fungi on Soil-Borne, Foliage
and Fruit-Damaging Phytopathogens
[0061] The effect of three basidiomycotine fungi, Meira
geulakonigii, Meira argovae and Acaromyces ingoldii on seven
phytopathogenic fungi, Colletotrichum gloeosporioides, Fusarium
mangiferae, Pencillium digitatum, Phytophthora citrophthora,
Rhizoctonia solani, Sclerotinia sclerotiorum and Sclerotium rolfsii
was assayed in the laboratory. The phytopathogens fungi were placed
on dialysis membranes covering media on which the basidiomycotine
fungi had formerly been cultured, thus exposing them to the
extracts of the latter. Acaromyces ingoldii inhibited the growth of
all phytopathogens fungi, whereas the Meira spp. hindered these
fungi to variable extents. Upon being returned to extract-free
media, all phytopathogenic fungi resumed growth, suggesting that
the inhibitory effect of the basidiomycotine fungi is fungistatic.
The three basidiomycotine fungi inhibited the growth of S.
sclerotiorum on tomato leaves for a few days and Meira spp. totally
hindered the infection of oranges by P. digitatum, whereas A.
ingoldii was less effective. Results of preliminary assays suggest
that neither competition for hydrocarbons nor secreted proteases
are responsible for the inhibitory activity of the basidiomycotine
fungi, whereas micro-molecules may play a part.
[0062] Interest in the biological control of phytopathogenic fungi
with microorganisms, including fungi, has increased in recent
years. We recently discovered several indigenous fungi that were
assigned to the Exobasidiomycetidae of the Ustilaginomycetes
(Basidiomycota), and described as new taxa by Boekhout, Theelen,
Houbraken, Robert, Scorzetti, Gafni, Gerson, and Sztejnberg (2003).
Although the fungi were obtained from cadavers of pestiferous
mites, we assayed one of these species, Meira geulakonigii
Boekhout, Gerson, Scorzetti & Sztejnberg, against a powdery
mildew. The rationale for this assay was the close phylogenetic
relationship of the genus Meira to Pseudozyma Bandoni emend.
Boekhout, one of whose species, Pseudozyma flocculosa (Traquair, L.
A. Shaw and Jarvis) Boekhout & Traquair, is known as an
antagonist of powdery mildews. The target disease was cucumber
powdery mildew, caused by Sphaerotheca fusca (Fr.) Blumer,
affecting cucumber. We found that leaf coverage of the powdery
mildew was significantly reduced and cucumber fruit yield was
significantly increased after being treated with blastoconodia of
M. geulakonigii (Sztejnberg, Paz, Boekhout, Gafni, and Gerson
2004). Later we reported that M. geulakonigii and the other new
species, Meira argovae Boekhout, Gerson, Scorzetti & Sztejnberg
and Acaromyces ingoldii Boekhout, Gerson, Scorzetti &
Sztejnberg, also inhibited two soil-borne phytopathogenic fungi,
Sclerotinia sclerotiorum (Lib.) de Bary and Sclerotium rolfsii
(Sacc.) (Gerson, Paz, Kushnir, and Sztejnberg 2005). We formerly
postulated that as neither Meira spp. nor A. ingoldii are parasitic
(Sztejnberg, Paz, Boekhout, Gafni, and Gerson 2004), their
antagonistic effect on mites and fungi is due to excreted toxins.
This hypothesis was confirmed upon exposing sclerotia of S.
sclerotiorum and S. rolfsii to crude extracts (e.g. devoid of
fungal matter) of all three fungi, and obtaining significant
inhibition of the growth of emerging mycelia (Gerson Paz, Kushnir,
and Sztejnberg 2005). In a later study (Paz, Burdman, Gerson, and
Sztejnberg 2007b) we found that, the crude extract of M.
geulakonigii secretions caused 100% mortality of a mite pest.
Herein we present data on the impact of these three fungal
biocontrol agents (FBAs) on phytopathogenic soil-borne fungi; we
add data about the effects of the secretions crude extract, from
the FBAs on S. sclerotiorum and S. rolfsii, as well as on
Rhizoctonia solani (Kuehn), the causative agent of root rot
diseases. We also describe the effects of the FBAs on pestiferous
leaf- and fruit-damaging fungi. These are: Colletotrichum
gloeosporioides (Penz.) Penz. & Sacc.; Fusarium mangiferae
Britz, Wingfield & Marasas; Pencillium digitatum Sacc. and
Phytophthora citrophthora (Sm. & Sm.) Leonian.
Source and Culture of the Beneficial Fungi
[0063] Details of the provenance of the FBAs are in Boekhout,
Theelen, Houbraken, Robert, Scorzetti, Gafni, Gerson, and
Sztejnberg (2003). Suffice to note that M. argovae (isolate AS005)
was obtained from cadavers of the two-spotted spider mite
(Tetranychus urticae Koch), M. geulakonigii (isolate AS004) from
dead citrus rust mites (Phyllocoptruta oleivora Ashmead), and A.
ingoldii (isolate AS001) was from the same mites at another site.
All isolates were routinely grown in the dark on 3.9% w/v potato
dextrose agar, amended with 250 ppm chloramphenicol (PDAC) at
25.degree. C., in Petri dishes (9 cm diam).
[0064] Meira argovae and M. geulakonigii were also grown within
Erlenmeyer flasks on the liquid medium potato dextrose broth (PDB),
but A. ingoldii (which does not form blastoconidia in liquid media)
was maintained on PDAC. Blastoconidia suspensions were obtained by
centrifuging (4,000 RPM) the contents of the flasks, pouring out
the supernatant and re-hydrating the blastoconidia-containing
residue with 50 cc deionized water (.sub.dH.sub.2O). The
concentration of the blastoconidia was then established by counting
with a h hemocytometer. Similar amounts of the blastoconidia of A.
ingoldii were obtained by adding five cc of .sub.dH.sub.2O to the
Petri dishes in which that fungus had developed and repeating the
same procedure.
Source and Culture of the Phytopathogenic Fungi and Chromista
[0065] A culture of S. sclerotiorum, which infects many commercial
crops, was obtained from Prof. O. Yarden of our university, from
the culture collection of the Department of Plant Pathology and
Microbiology at the Hebrew University of Jerusalem, Israel. This
fungus secretes oxalic acid that damage plants and forms sclerotia
that may survive in the soil for long periods (Agrios 2005).
Cultures of S. rolfsii and of R. solani, both of which also infect
many crops, were obtained from Prof. Y. Katan, also of our
university; these fungi also form sclerotia. They were maintained
on PDAC. Prior to exposing them to the various crude extracts they
were grown on DWA (deionized water agar) at their preferred
temperatures (S. sclerotiorum at 19.degree. C., S. rolfsii and of
R. solani at 29.degree. C.). Sclerotia of the fungi were obtained
by letting their cultures dry up and then harvesting the dried
sclerotia. Cultures of C. gloeosporioides and of F. mangiferae were
obtained from Dr. S. Freeman, Volcani Institute, Bet Dagan, Israel.
The culture of P. digitatum was received from Dr. S. Droby at the
same institute, and that of P. citrophthora from our lab culture
collection. Colletotrichum gloeosporioides damages many field crops
as well as citrus and avocado, whereas F. mangiferae is the
causative agent of mango malformation disease. Pencillium digitatum
causes the green mould that damages picked citrus fruit (e.g. a
post-harvest disease), and P. citrophthora affects the citrus by
causing food rot, gummosis and fruit brown rot. All fungi cultured
like the FBAs. Prior to exposing them to the various extracts they
were transferred from PDA to DWA (in order to avoid the residual
effects of nutrients that could interfere with extract effects) and
incubated at 25.degree. C.
[0066] Short-term, non-replicated observations were also conducted
on two additional pathogenic fungi, namely Alternaria alternate
(Fr.) Keissl, obtained from our University, and Botrytis cinerea
(De Bary) Whetzel from Dr. Y. Elad, Volcani Institute, Bet Dagan,
Israel. The former causes leaf spots on many commercial plants,
whereas the latter causes grey mould on grape, strawberry and
various vegetables crops. They were cultured as above.
Experimental Set-Up
[0067] In order to eliminate any accidental contact between the
FBAs and the pathogens, discs (18 mm diam) of each of the former,
taken from one-week old colonies, were transferred onto membranes
cut from dialysis tubing 12-kDa cutoff (Visking dialysis membrane,
12 kDa, Medicell International LTD, London, UK) placed on PDAC in
Petri dishes. The area of the membranes was larger than the dishes,
covering the medium as well as the inner walls of the dishes, in
order to avoid any contact between media and fungi. These membranes
are known to allow the diffusion of micro-molecules but not of
macro-molecules or enzymes. The dishes were incubated at 25.degree.
C. for 10 days, the membranes with the FBAs then being removed,
leaving the media with the extracts of each FBA without any
residues of their mycelia or blastoconidia ("extract dishes"
hereinafter). Discs (eight mm diam) of the pathogen cultures
(including the two "short-term" fungi), which had been maintained
on DWA, were then placed in the center of the extract dishes.
[0068] Most cultures (except S. sclerotiorum at 19.degree. C., S.
rolfsii and of R. solani at 29.degree. C.) were kept at 25.degree.
C. and their mycelial growth was measured every 24 hrs; the
obtained data were used for estimating colony growth. Each
pathogen-FBA combination was replicated eight times, in two series.
Control dishes (with PDAC only) were inoculated with the same
pathogens and similarly examined. After recording the effects of
the extract, all pathogen-bearing discs were placed onto PDAC
plates without any FBA extracts. This was done in order to observe
any further fungal growth and thus determine whether the extract
effects were fungicidal or fungistatic.
[0069] The amount of conidia or sclerotia produced by each pathogen
(except P. citrophthora, a species that produces zoospores, which
cannot be assayed by this method) was estimated by adding five cc
of .sub.dH.sub.2O to each plate, pouring the obtained suspense into
an Eppendorf tube and then counting with a hemacytometer. Number of
replicates was as above.
Germination and Production of Pathogen Sclerotia and Conidia
[0070] The effect of the FBAs on the germination and mycelial
growth from dried sclerotia was assayed by placing 20 sclerotia of
S. rolfsii and 20 of S. sclerotiorum on the extract dishes obtained
from each FBA (R. solani did not form sclerotia in our cultures),
and measuring colony growth, as above. The dishes with S. rolfsii
were kept at 29.degree. C., those of S. sclerotiorum at 19.degree.
C., and extent of germination was assessed after two days.
Production of sclerotia by S. rolfsii and by S. sclerotiorum in the
pathogen cultures kept on the extract dishes was monitored after 15
and 10 days, respectively. Due to the fact that S. sclerotiorum
produced no sclerotia (see below), only the sclerotia of S. rolfsii
were tested for viability by placing them on filter paper
impregnated with bromo cresol blue, which changes its color to
dark-yellow as the sclerotia germinate (Gamliel, Grinstein, Klein,
Cohen, and Katan 1998).
[0071] Suspensions of conidia (10.sup.8 ml.sup.-1) in
.sub.dH.sub.2O of C. gloeosporioides, F. mangiferae and P.
digitatum were placed on the extract dishes of the FBAs and
incubated at 25.degree. C., the former two for one week, P.
digitatum only during four days. At the end of these periods, the
number of germinating conidia (whose germ tubes exceeded the length
of the pertinent conidium) was counted in four microscopic fields
per dish, averaged and percent of germination was calculated. Each
pathogen-FBA combination was replicated four times, in two series.
Control dishes were prepared and examined as above.
[0072] In addition, discs with the conidia of P. digitatum, which
did not germinate at all (see below), were transferred onto PDAC
dishes devoid of any fungal extracts, in order to determine the
duration of the inhibition, as above.
Inhibition of Sclerotinia sclerotiorum by the Beneficial Fungi on
Tomato Leaves
[0073] Young tomato leaflets, placed on humid filter paper in Petri
dishes, were sprayed with 1-5.times.10.sup.8 ml.sup.-1 suspensions
of the blastoconidia of each of the FBAs in .sub.dH.sub.2O; control
leaves were treated with water only. The closed dishes were kept
for 10 days at 19.degree. C., after which discs (2 mm diam) taken
from the middle of a S. sclerotiorum culture were placed on each
leaflet. The dishes were returned to 19.degree. C. Mycelial growth
on the leaves was measured every 24 hrs (until the control leaves
became covered by the pathogen), and the obtained data were used
for estimating the extent of FBA inhibition. Each FBA was assayed
twice, each time on 5-8 leaflets, in 5 replicates.
[0074] Tomato leaves treated with the FBA and then with S.
sclerotiorum were examined by a scanning electron microscope (SEM)
as previously described (Paz, Burdman, Gerson, and Sztejnberg
2007b). In short, samples were immersed in glutaraldehyde (5%) for
two hours, washed thoroughly with buffer phosphate (pH 7.2), dried
and gold-plated. They were then examined by a JSM 5410 scanning
electron microscope (Jeol Ltd, Tokyo, Japan), in high vacuum mode,
in order to examine the actual interactions between the FBAs and
the pathogen on the leaves.
Inhibition of Citrus Green Mould of Oranges by the Beneficial
Fungi
[0075] Sweet oranges (Citrus sinensis cv. New Hall) picked from an
organic orchard near Rehovot were surface sterilized with 90%
ethanol. FBA blastoconidia were obtained as above, and a suspension
(10.sup.8 ml.sup.-1) of each was sprayed onto the oranges, which
were then kept in a sterile humid chamber at 25.degree. C. for 10
days. At that time a small wound (2 mm length and depth) was
scratched on its peel by a sterilized instrument, and 50 ml of the
P. digitatum conidial suspension (5.times.10.sup.3 ml.sup.1) was
smeared on each wound. Each FBA was applied onto 10 oranges (in two
series of five each), which were kept, along with suitable controls
(wounded but untreated with FBAs) at 25.degree. C. for observation.
Extent of damage was estimated when the control oranges were
completely covered by the mould. Thin cuttings were taken from the
oranges that showed green mould inhibition and prepared for SEM
examination, as above.
Examining the Mode of Action of the FBAs
[0076] Several tests were run in order to obtain preliminary data
on the inhibitory mode of action of the FBAs. The tests included
determining their rate of hydrocarbon consumption, evaluating the
effect of micro-molecules (which are known to traverse the dialysis
membrane, in contrast to macro-molecules and enzymes), and
determining whether lytic proteases of FBA origin inhibit the
pathogens.
Utilization of Hydrocarbons (Sugars)
[0077] This test was run to determine whether the inhibitory
activities of the FBAs were due to competition for carbohydrates.
It was conducted by using the slightly-modified procedure of Poola,
Bhuiyan, Ortiz, Savant, Sidhom, Taft, Kirschenbaum, and Kalis
(2002). The Anthrone reagent was prepared by adding 70 ml of
concentrated sulfuric acid to 30 ml of .sub.dH2O and then 200 mg
Anthrone (Sigma). A standard (or dilution) curve was prepared by
placing 0.0, 0.005, 0.01, 0.05 and 0.1 mg glucose to one ml of
.sub.dH.sub.2O in glass tubes and adding 5 ml Anthrone to each
tube. The tubes were then boiled in a water bath of 100.degree. C.
for five minutes, and one ml from each tube glucose concentration
was then read at 620 nm by a spectrophotometer in order to obtain a
standard curve. Media that had been covered by the dialysis
membrane, on which the FBAs had grown, were tested after 10 days.
Discs (13 mm diam.) were taken from each FBA (from three different
extract dishes) and placed into plastic sealed tubes (13 ml). The
media in the tubes were liquidized in boiling water, and samples of
0.1 ml from each tube were diluted in 0.001, 0.005 and 0.01
.sub.dH.sub.2O. From each diluted one ml was transferred to a glass
tube to which five ml of the Anthrone reagent were added, and
maintained for five minutes in a water bath of 100.degree. C. One
ml from each sample was transferred to a cuvette and read in a
spectrophotometer at 620 nm. Four separate readings were done for
each dilution and two separate experiments were conducted for each
FBA. The rate of sugar utilization by the FBAs was calculated by
comparing the obtained results with data from the dilution
curve.
Effect of Proteases Secreted by the FBAs
[0078] These tests were conducted to determine whether the
inhibitory activities of the FBAs were due to their secreted
proteases (Elad and Kapat 1999). The experimental procedure was
slightly modified from Kanemitsu, Nishini, Kunishima, Okamura,
Takemura, Yamamoto, and Kaku (2001), were conducted in order to
determine if lytic proteases of FBA origin, whether in the extract
dishes or originating from FBA blastoconidia, participate in
pathogen inhibition. The experiments were run in Petri dishes
containing five ml gelatin as the substrate. Discs (eight mm diam)
from 10-days-old FBA cultures that had been covered by the dialysis
membranes were placed in the centre of the gelatin in the dishes
and incubated for 7 days at 25.degree. C. Protease activity was
determined by adding a 0.1 ml solution of 15% trichloroacetic acid
(TCA) to each dish. TCA causes the gelatin becoming murky whereas
the broken-down substrate becomes transparent; a clear halo thus
forms in the middle of the dishes. Discs from pristine (devoid of
FBAs) PDA, and similar discs to which 0.02 ml of proteinase K
(Sigma) from a concentration of 0.1 gr/ml dH.sub.2O had been added,
served as controls, and protease activity was measured as
above.
[0079] Tests of proteases secretion by blastoconidia of M. argovae
and M. geulakonigii was done in Petri dishes containing five ml
gelatin served as substrates. Small wells (3.0 mm depth, 8.0 diam)
were bored in the middle of the gelatin in each dish. PDB media on
which M. argovae and M. geulakonigii had developed (as noted, A.
ingoldii forms no blastoconidia in a liquid medium) were
centrifuged for 10 min at 25.degree. C. at 4,000 rpm. A small
sample (0.2 ml) from the supernatant (of each FBA), containing the
blastoconidia that had not precipitated, was then placed in the
wells. Another sample was filtered through a sieve (0.45 micrometer
holes) (Schleicher & Schull, London) thus obtaining
blastoconidia-free liquid, which was also placed in the gelatin
wells. There were two controls: pristine PDB was placed in one
group of wells, and proteinase K (0, 02 ml) into another. All
dishes were incubated for three days at 25.degree. C. and assessed;
each FBA was assayed in two series, each with four replicates.
Effect of Micro-Molecules Produced by the FBA. The purpose of this
test was to determine whether the inhibitory activities of the FBAs
were due to secreted micro-molecules, which could have traversed
through the 12 kDa dialysis membrane, or to other compounds,
tentatively called toxins. This necessitated a two-stage assay:
first applying the heated purified active material on a bacterial
colony, and then the heated contents of extract dishes on the
pathogenic fungi.
[0080] The test bacterium was Agrobacterium tumefaciens (strain
ID1), obtained from Prof. Leonid Chernin of our University already
shown to be susceptible to the FBAs. Agrobacterium tumefaciens was
grown overnight in tubes containing five ml Lennox Broth (LB)
(Difco) undergoing constant shaking at 28.degree. C. A 50-.mu.l
aliquot of this bacterial suspension was added to five ml of LB
containing 0.6% agar, and placed into small (five mm diam) Petri
dishes. Apparent active toxins, obtained from the extract dishes of
10-days' old FBA colonies, were purified using HPLC procedures (Paz
2007) and heated to 100.degree. C. for 15 min. Ten .mu.l of the
heated medium from each FBA were then chilled and poured into the
middle of the A. tumefaciens dishes. Similar amounts of the
purified but unheated toxins, as well as unheated media from the
extract dishes of each FBA, served as controls. Dishes were
incubated at 28.degree. C. for 24h and observed for any developed
inhibition haloes.
[0081] The effect of FBA proteins (micro-molecules) secreted into
the media was assayed by taking discs (13 mm) from 10 days-old FBA
colonies, sealing them in sterile tubes (13 ml), and immediately
placing them in boiling water for 15 min, until complete boiling of
the media. The boiled media were poured into 5 ml Petri dishes and
after solidification DWA discs of one-week old cultures, of either
P. digitatum or S. sclerotiorum were placed at the middle of the
dishes. Non-boiled media from the FBAs served as controls. The P.
digitatum dishes were maintained for three days at 25.degree. C.
and those of S. sclerotiorum at 19.degree. C. All experiments were
repeated twice for each pathogen, in four replicates (tubes).
Statistics Analyses
[0082] All obtained data of growth and germination inhibition were
analyzed using two-way ANOVA by Dunnett's test (p=0.05), using JMP
5.1.2. Software (SAS Institute, Cary, N.C.). The data were arc-sin
transformed prior to analysis. The data obtained from the control
treatments were used as reference groups.
Results--Inhibition of Colony Growth and of Conidia Production
[0083] These experiments had to be terminated after a few days (3-4
with R. solani, S. rolfsii and S. sclerotiorum, about a week with
the other fungi) because the control dishes were totally overrun by
the pathogen mycelia. The FBAs inhibited the growth of all
pathogens, but the pattern of inhibition differed. Strongest
inhibition was obtained in the extracted dishes on which A.
ingoldii had grown (FIG. 1); only R. solani and F. mangiferae
producing any mycelia thereon. The extracts of M. argovae and M.
geulakonigii also inhibited all pathogens, but to a variable
extent. They had a similar inhibitory effect on P. digitatum and on
P. citrophthora, affected C. gloeosporioides, F. mangiferae (which
was totally unaffected by the M. geulakonigii extracts) and R.
solani to different degrees and had a similar effect on S.
sclerotiorum; S. rolfsii appeared to be the least affected.
[0084] The short-term observations showed that the extracts of the
FBAs also inhibited the growth of the A. alternata and B. cinerea
colonies. The effect on the former was rather mild, similar to that
C. gloeosporioides, whereas the latter was more strongly affected,
like S. sclerotiorum.
[0085] When the discs with the hitherto-inhibited pathogen cultures
were subsequently placed on PDAC without any FBA extracts, all
fungi resumed growth and production of conidia.
Inhibition of Sclerotia and Conidia Production
[0086] No sclerotia were produced by S. sclerotiorum growing on the
extract dishes of the three FBAs. Sclerotium rolfsii, in contrast,
produced a variable number (an average of 39.+-.9.0 on A. ingoldii,
87.+-.9.3 on M. argovae and 50.+-.5.2 on the M. geulakonigii
extracts, all significantly less than in the control dishes, which
came to an average of 343.+-.19.3). Also, its sclerotia grew only
on the dishes' margins and were clearly smaller than those in the
controls. The viability (as tested with bromocresol blue) of the S.
rolfsii sclerotia that had developed on the extracts of the FBAs
did not differ from that of the control sclerotia, in all cases
coming to nearly 100%.
[0087] Pencillium digitatum that had developed on the extract
dishes of A. ingoldii formed no conidia at all, but produced
similar amounts (about 5.times.10.sup.2) on the two Meira spp. The
number of conidia formed by Fusarium mangiferae and C.
gloeosporioides did not differ from the amount produced in the
controls (data not shown).
[0088] The non-replicated observations on B. cinerea showed that
this pathogen was inhibited by the FBAs to the same extent as S.
sclerotiorum. The development of A. alternata was hindered to a
lesser degree, similar to that seen with C. gloeosporioides.
Germination of Sclerotia and of Pathogen Conidia
[0089] The germination of the sclerotia of S. sclerotiorum and S.
rolfsii on the various FBAs extracts differed between the two
pathogens. The extracts of M. argovae and M. geulakonigii had no
effect on sclerotial germination of the former pathogen, whereas
those of A. ingoldii totally inhibited germination. However, the
germination of the sclerotia of S. rolfsii on the extracts of the
latter pathogen was affected by their number per dish. None
germinated when only one was placed per dish, but 70% and 82%
germinated, respectively, when 5 or 20 were put together (Table
1).
TABLE-US-00001 TABLE 1 Percent germination of the sclerotia of
Sclerotium rolfsii on extract dishes on which Acaromyces ingoldii
(Ai), Meira argovae (Ma) and by Meira geulakonigii (Mg) had
developed. No. sclerotia Ai Ma Mg Control 1 0 100 75 100 5 70 100
100 100 20 82 100 100 100
[0090] Conidial germination of P. digitatum was completely
inhibited by all three FBAs, whereas M. argovae had no effect on C.
gloeosporioides and on F. mangiferae, and M. geulakonigii affected
only the latter pathogen (Table 2). When conidia of P. digitatum,
which had hitherto failed to germinate, were placed on the
extract-free PDAC, they germinated and formed normal mycelia.
TABLE-US-00002 TABLE 2 Percent germination of the conidia of C.
gloeosporioides, F. mangiferae and P. digitatum on extract dishes
on which Acaromyces ingoldii (Ai), Meira argovae (Ma) and by Meira
geulakonigii (Mg) had developed. Asterisk indicates significant
differences from the control at P < 0.05. Pathogen Ai Ma Mg
Control Colletotrichum gloeosporioides 1* 93 92 93 Fusarium
mangiferae 10* 90 46 93 Pencillium digitatum 0 0 0 85
Inhibition of Mycelial Growth from the Sclerotia
[0091] Strongest inhibition was again caused by the extract dishes
on which A. ingoldii had grown, as no mycelia of S. sclerotiorum or
of S. rolfsii developed thereon. The former pathogen's growth was
also almost completely inhibited by the M. argovae and the M.
geulakonigii extracts (FIG. 9A). In contrast, colony growth of S.
rolfsii, although still significantly less than in the controls
(FIG. 9B), was substantial on the M. argovae and M. geulakonigii
extract dishes. The growth pattern of S. rolfsii from its sclerotia
on the extracts was similar, but slower, to its development from
mycelia (FIG. 1).
Inhibition of S. sclerotiorum by the FBAs on Tomato Leaves
[0092] All FBAs significantly inhibited the growth of S.
sclerotiorum on the tomato leaflets, albeit to a different extent
(FIG. 10). However, by the fourth day the severity of the
inhibition declined. Later observations indicated that all leaflets
were subsequently overrun by the pathogen (data not shown).
Inhibition of Green Mould Inoculation of Oranges by the FBAs
[0093] Meira argovae and M. geulakonigii completely inhibited the
development of green mould on the infected oranges, whereas the
effect of A. ingoldii was far less benign (50% infection), although
the damage was still significantly less than in the control fruit
(80% infection) (FIG. 11).
[0094] The SEM micrographs show that all three FBAs were present on
the fruits peel and penetrated it via the stomata (FIG. 12).
Utilization of Carbohydrates (Sugars)
[0095] Sugar utilization by the FBAs was quite low, as its
depletion by A. ingoldii after 10 days came to 15.6% (as compared
to the controls), by Meira argovae to 14.5% and by M. geulakonigii
to 21.9%. This suggests that competition for hydrocarbons is, at
most, a minor factor affecting pathogen inhibition by the FBAs.
Effect of Proteases Secreted by the FBAs
[0096] Weak haloes of ca 1-2 mm developed after one-two weeks of
incubation in the dishes containing the FBA mycelia, whereas no
haloes appeared in dishes with PDB (data not shown). This indicates
the production of external protease by the mycelia of both FBAs
assayed. On the other hand, no haloes (meaning no protease
secretion) were seen in the assays with the blastoconidia presence
suggesting that the latter do not produce proteases.
Effect of Micro-Molecules Produced by the FBA.
[0097] The denaturation of the micro-molecules--caused by the
boiling water bath--significantly decreased the inhibitory effect
of M. argovae and of M. geulakonigii on S. sclerotiorum and P.
digitatum, as compared with the controls (FIG. 6). However,
considerable, significant inhibition remained. On the other hand,
the boiling treatment did not reduce the inhibitory activity of the
A. ingoldii extract plates, indicating that different secondary
compounds were secreted by this fungus.
[0098] Boiling the protein micro-molecules produced by M. argovae
and M. geulakonigii had no inhibitory effect on the rate of growth
of A. tumefaciens, as similar haloes of inhibition developed in the
microbial colonies exposed to the boiled and to the non-boiled
extracts (data not shown).
[0099] Fungi used in the biological control of plant pathogens use
a variety of antagonistic methods, including parasitism,
competition for living space and for nutrient sources, changing the
chemical environment, the secreting of toxic or inhibiting
secondary compounds and compounds that induce the production of
host-plant resistance factors (Agrios 2005; Elad 2000). The fact
that all pathogens which were placed on pristine PDAC, after
exposure to the FBA extracts, resumed normal growth, strongly
suggests that the adverse effect of the FBAs is probably
fungistatic. This hypothesis is supported by the observation that
conidia of P. digitatum that had hitherto failed to develop on the
extract dishes, germinated and formed normal mycelia when placed on
extract-free PDAC, and by the short-term effect of the FBAs on S.
sclerotiorum infecting tomato leaves. In addition, the inhibitory
effect of the FBA extracts appears to be dosage-dependent, as seen
by the decline of inhibition when more sclerotia were placed in a
dish.
[0100] Many antagonistic fungi inhibit the development of pathogens
by competition for nutrients; for instance, Ulocladium atrum
(Preuss) Sacc. is known to utilize available dietary resources
faster than S. sclerotiorum, and thereby to control the latter. Our
preliminary data on the possible modes of action of the FBAs
indicate that competition for sugars most likely does not play a
role in the inhibition of pathogen growth. It is known that some
antagonistic fungi secrete proteases in order to harm pathogens;
for instance, Trichoderam harzianum Rifai secretes proteases that
reduce the activity of the pathogen B. cinerea. As to the FBAs, a
rather minor role may at best be allocated to the excretion of
proteases. As to macro-molecules, boiling suggests that they are
likewise not involved, but micro-molecules cannot be ruled out,
because they decreased the inhibitory effect of the two Meira spp.
(FIG. 13).
[0101] We formerly noted (Paz, Gerson, and Sztejnberg 2007a) that
each of the three FBAs had a variable effect on several pestiferous
mite (Acari) species, and a similar phenomenon was seen in this
study. Each of the FBAs affected every pathogen differently,
indicating a distinct degree of selectivity which was probably due
to their different toxins. The extracts of A. ingoldii had the
strongest effect on all target-fungi, whereas the influence of the
two Meira spp. was variable. It is however of interest that whereas
the extracts of A. ingoldii totally inhibited the colony growth P.
digitatum (FIG. 8), this FBA afforded oranges significantly less
protection against the pathogen than the Meira spp. (FIG. 11). Such
differences in the effectiveness of FBAs when applying them in the
laboratory or in the field are well known.
[0102] The penetration of orange stomata (FIG. 12) suggests that,
as with grapefruits (Paz, Burdman, Gerson, and Sztejnberg 2007b)
the FBAs may live within that fruit's tissues (e.g. are
endophytic). The long-term influence of these endophytic FBAs
(whose effect, as noted, is mostly fungistatic) is probably due to
their continuous secretion of the inhibiting toxins. Backman and
Sikora (2008) noted endophytes as an emerging tool for biocontrol,
which is consistent with our finding of orange green mould
inhibition by the FBAs (especially by Meira spp.) (FIG. 11). These
micrographs (FIG. 12) suggest that all three FBAs may have an
affinity for the conidia and germ tubes of P. digitatum, causing
their partial collapse. Subsequent experiments may show whether
inoculating citrus fruit with the FBAs could serve as a defense
mechanism against fungi.
[0103] Formerly we showed that the three FBAs inhibited several
species of plant-feeding mites (Gerson, Paz, Kushnir, and
Sztejnberg 2005; Paz, Gerson, and Sztejnberg 2007a), and now we
broaden this spectrum of susceptible target-pests to
phytopathogenic fungi.
[0104] Finally, it is of interest that the pathogens, which are
assigned to two different Kingdoms: Kingdom Fungi and Kingdom
Chromista (Agrios 2005). The former includes taxa of different
higher fungal groupings (C. gloeosporioides, F. mangiferae, P.
digitatum and. S. sclerotiorum in the Ascomycotina, R. solani and
S. rolfsii in the Basidiomycotina. Phytophthora citrophthora, on
the other hand, belongs to the Oomycotina in the Kingdom
Chromista). Thus it is noteworthy that all were similarly affected
by A. ingoldii (FIG. 8), whereas the effect of the two Meira spp.
on the seven pathogens was variable. In addition, the Basimycotine
S. rolfsii was less hindered by the Meira spp. than were the two
Ascomycotina pathogens (FIG. 8 and FIG. 9B), suggesting a variable
susceptibility to the FBAs. Finally, the wide range of pathogenic
fungi that were susceptible to the FBAs suggests that other
pathogens (including pathogenic bacteria) may also be inhibited by
the FBAs.
[0105] While this invention has been described in terms of some
specific examples, many modifications and variations are possible.
It is therefore understood that within the scope of the appended
claims, the invention may be realized otherwise than as
specifically described.
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