U.S. patent application number 12/799305 was filed with the patent office on 2010-11-18 for species of fungi and their use in pest and disease control.
This patent application is currently assigned to YISSUM RESEACH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM. Invention is credited to Aviva Gafni, Uri Gerson, Abraham Sztejnberg.
Application Number | 20100291135 12/799305 |
Document ID | / |
Family ID | 27590151 |
Filed Date | 2010-11-18 |
United States Patent
Application |
20100291135 |
Kind Code |
A1 |
Sztejnberg; Abraham ; et
al. |
November 18, 2010 |
Species of fungi and their use in pest and disease control
Abstract
The present invention describes substantially pure cultures of
three new species of fungi, Meira geulakonigae (CBS 110052), Meira
argovae (CBS 110053) and Acaromyces ingoldii (CBS 110050) (all nom.
prow.), wherein said cultures are biologically pure. The
conditioned medium for all three new fungi species is also
described, as well as their biologically active products. The
biological product of the three new fungi species and their
conditioned medium has miticidal and fungicidal activities. Thus, a
composition for the control of mites and/or phytopathogenic fungi
comprising a biocontrol agent derived from any one of the three new
fungi species is also described, optionally comprising an
agriculturally compatible carrier. Finally, the present invention
describes a method for producing a fungicide and/or a miticide,
wherein the active ingredient of said fungicide/miticide is any one
of M. geulakonigae, M. argovae, or A. ingoldii, or any combination
thereof, and/or a culture thereof, and/or any product or derivative
thereof, comprising culturing said fungus and optionally isolating
said active ingredient from the culture.
Inventors: |
Sztejnberg; Abraham;
(Rehovot, IL) ; Gerson; Uri; (Rehovot, IL)
; Gafni; Aviva; (Rishon LeZion, IL) |
Correspondence
Address: |
COOPER & DUNHAM, LLP
30 Rockefeller Plaza, 20th Floor
NEW YORK
NY
10112
US
|
Assignee: |
YISSUM RESEACH DEVELOPMENT COMPANY
OF THE HEBREW UNIVERSITY OF JERUSALEM
|
Family ID: |
27590151 |
Appl. No.: |
12/799305 |
Filed: |
April 22, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10502486 |
Feb 10, 2005 |
7731984 |
|
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PCT/IL03/00059 |
Jan 23, 2003 |
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12799305 |
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Current U.S.
Class: |
424/195.15 ;
800/295 |
Current CPC
Class: |
C12N 1/14 20130101; C12R
1/645 20130101; Y10S 435/911 20130101; A01N 63/30 20200101 |
Class at
Publication: |
424/195.15 ;
800/295 |
International
Class: |
A01N 65/00 20090101
A01N065/00; A01P 3/00 20060101 A01P003/00; A01H 15/00 20060101
A01H015/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 24, 2002 |
IL |
147835 |
Claims
1. A substantially pure culture of the fungus Meira geulakonigae
nom. prov. (M. geulakonigae).
2. The culture of claim 1, wherein said culture is biologically
pure.
3. The culture of claim 1, wherein said fungus is the fungus
designated CBS Accession No. 110052.
4. A conditioned medium of the fungus of claim 1.
5. A substantially pure culture of the fungus Meira argovae nom.
prov. (M. argovae).
6. The culture of claim 5, wherein said culture is biologically
pure.
7. The fungus of claim 5, designated CBS Accession No. 110053.
8. A conditioned medium of the fungus of claim 5.
9. A substantially pure culture of the fungus Acaromyces ingoldii
nom. prov. (A. ingoldii).
10. The culture of claim 9, wherein said culture is biologically
pure.
11. The fungus of claim 9, designated CBS Accession No. 110050.
12. A conditioned medium of the fungus of claim 9.
13. Biologically active products derived from fungi selected from
the group consisting of M. geulakonigae, M. argovae and A.
ingoldii.
14. The biologically active products of claim 13, wherein the
biological activity of said products is miticidal and/or
fungicidal.
15-23. (canceled)
24. A composition for the control of fungi, wherein said
composition comprises at least one biocontrol agent, derived from
any one of the fungi M. geulakonigae, M. argovae, or A. ingoldii,
and/or from a product thereof, optionally further comprising an
agriculturally compatible carrier.
25. The composition of claim 24, wherein said biocontrol agent is
spores and/or products thereof.
26. The composition of claim 25, wherein said carrier is extracted
from a liquid culture of any one of said fungi.
27. The composition of claim 26, wherein said carrier is selected
from the group consisting of water, culture medium, oils or other
adjuvants or carriers.
28. The composition of claim 27, in the form of liquid suspension
containing between about 10.sup.7 to about 10.sup.10 spores/ml.
29. The composition of claim 28, wherein the suspension contains
about 10.sup.8 spores/ml.
30. The composition of claim 24, further comprising a conventional
adjuvant used in formulations for biocontrol agents.
31. A method for producing a fungicide and/or a miticide, wherein
the active ingredient of said fungicide/miticide is any one of M.
geulakonigae, M. argovae, or A. ingoldii, or any combination
thereof, and/or a culture thereof, and/or any product or derivative
thereof, comprising culturing said fungus and optionally isolating
said active ingredient from the culture.
32. The method of claim 31, wherein said method comprises the steps
of: (a) cultivating, under aerobic or anaerobic conditions, any one
of the fungi M. geulakonigae, M. argovae, or A. ingoldii in an
appropriate culture medium; (b) harvesting the culture; (c)
separating the spores from the liquid medium; and either (d)
suspending the spores in an agriculturally compatible liquid
carrier to give a fungicide/miticide in liquid form; or (e)
lyophilizing the spores, to give a dry form fungicide/miticide.
33. The method of claim 32, wherein said dry form
fungicide/miticide is further dissolved in an agriculturally
compatible liquid carrier.
34. The method of claim 31, wherein the fungus is cultured at a
temperature of from about 20.degree. C. to about 30.degree. C.
35. The method of claim 34, wherein the temperature is from about
23.degree. C. to about 27.degree. C.
36. The method of claim 35, wherein the temperature is 25.degree.
C.
37. The method of claim 31, wherein the fungus is cultured at a pH
from about 5.1 to about 5.6.
38. The method of claim 37, wherein the pH is about 5.1.
39-41. (canceled)
42. A method of controlling fungi contamination in a plant
susceptible to said fungi, which comprises applying the fungicide
composition of claim 24 to the plant.
43. The method of claim 42, wherein the fungal infestation is due
to Sphaerotheca fusca (powdery mildew).
44. The method of claim 43, wherein said plant is any commercial
plant susceptible to powdery mildew diseases or other
phytopathogenic fungal diseases.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to three new species of fungi
of the Subdivision Basidiomycotina, Order Ustilaginales, to any
products or other chemicals obtained therefrom or from their
progeny and/or any mutants thereof, and to their use in the control
of pests, particularly pestiferous mites (Acari) and pathogenic
plant fungi, especially powdery mildews.
BACKGROUND OF THE INVENTION
[0002] Plant diseases caused by fungi and mites have a significant
adverse impact on the production of important crops worldwide.
[0003] Pest mites belong to two main families. The most important
family is Tetranychidae, also known as the spider mites. These
mites cause widespread damage to almonds, apple, avocado, cassava,
citrus, cotton, grapes, tea, various greenhouse crops like
cucumber, pepper and tomato, and several ornamentals, including
roses and chrysanthemums [Helle, W. & Sabelis, M. W. Spider
Mites, Their Biology, Natural Enemies and Control, Volumes. I and
II, Elsevier, Amsterdam (1985)]. The other family, Eriophyidae,
corresponds to the rust or gall mites, which can be major pests of
apple, citrus, coconut, pear, currants and berries, as well as many
vegetables and ornamentals [Lindquist et al: (Eds.) Eriophyoid
Mites, Their Biology, Natural Enemies and Control. Elsevier,
Amsterdam (1996)]. The carmine spider mite, Tetranychus
cinnabarinus, and the citrus red mite, Panonychus citri, belong to
the former family, whereas the citrus rust mite, Phyllocoptruta
oleivora, to the latter. In Israel, the carmine spider mite is an
important pest of vegetables and ornamentals, whose chemical
control has met increasing difficulties due to evolving resistance
to pesticides. The citrus red mite is of minor importance, whereas
the citrus rust mite is the major pest of citrus in Israel and many
other parts of the world. Local citrus growers have been
encountering serious problems in the chemical control of the citrus
rust mite, due to its increased resistance to acaricides and the
presence of pesticide residues on exported fruit [Palevsky et al.
Mite problems on citrus and control strategies in Israel.
Proceedings of the International Congress of Citriculture, (in
press)]. In counterpart, biological control of this pest with
introduced predators has just begun and is still uncertain [Argov,
Y., S. Amitai, G. A. C. Beattie and U. Gerson. Rearing, release and
establishment of imported predatory mites to control citrus rust
mite in Israel. BioControl, 47: 399-409 (2002)].
[0004] Likewise, fungal phytopathogens cause major and severe
economic losses in the agricultural and horticultural industries.
Germination and growth of infected plants are inhibited; plants
succumb to soft rot and may even die. Infested soils are unsuitable
for growing certain crops. The powdery mildews are the most common,
widespread and easily recognizable plant diseases. They are caused
by fungi of the family Erysiphaceae, which are grouped into seven
main genera [Agrios, G. N. Plant Pathology, 4th ed. Academic Press,
New York (1997); Braun, U. A Monograph of the Erysiphales (Powdery
Mildews), J. Cramer, Grebruder Borentraeger, Berlin (1987)].
Powdery mildews are characterized by spots or patches of a white to
grayish powdery growth on young plant tissues, which can cover
entire leaves and other organs of the plant [Agrios (1997) id
ibid.]. Powdery mildews are very often present on crop plants and
ornamentals, and the yield losses that they incur annually on all
crops probably surpasses the losses caused by any other single type
of plant disease. Crops that suffer severely from powdery mildews
include wheat, barley, cucumber, squash, strawberry, grape, apple,
mango, ornamentals such as rose, begonia, lilac and many others
[Agrios (1997) id ibid.]. Chemical fungicides usually control these
diseases, but this mode of control is continuously being challenged
by the rapid development of resistance to the recommended
fungicides. As for the mites pest, biological control of fungal
phytopathogens is just in its infancy as well. Although the
biofungicide AQ10 has been registered for powdery mildews control
since 1996, the extent of its use is still moderate when compared
to the use of chemical fungicides [Copping, L. G. (Ed.) The
BioPesticide Manual, 1.sup.st ed. British Crop Protection Council,
Farnham (1998); Sztejnberg et al. Ampelomyces quisqualis for
biological and integrated control of powdery mildews in Israel. J.
Phytopathology 124:285-295 (1989)]. Overall, world expenditure for
fungicides to control powdery mildews is estimated to be circa one
billion US dollars per year.
[0005] In summary, plant mites and fungal phytopathogens are still
mostly controlled with chemical acaricides and fungicides, despite
the above-mentioned corollary problems. Thus, the widespread abuse
of pesticides and the emergence of pest resistance to most of these
chemicals has increased the demand for environmentally-friendly
biological pesticides, or "biopesticides" [Copping, 1998, id
ibid.].
[0006] In order to fulfill this demand, it is a major object of the
invention to provide new species of fungi that possess activity
against plant mites and fungi, and that can be used against them.
Another object of the invention is to provide anti-fungal and
anti-miticidal preparations, or biopesticides.
[0007] So far, there has been no indication that any of the three
new fungal species hereby described and claimed, have had any
adverse effect on the plants with which they were in contact in any
way, nor were they detrimental to any other organism (except the
pests being assayed against). It is therefore a further object of
the invention to provide miticidal and fungicidal compositions that
would be devoid of adverse effects on the treated plants, or on the
environment.
[0008] These and other objects of the present invention will become
apparent as the description proceeds.
SUMMARY OF THE INVENTION
[0009] The present invention relates to three new species of fungi
of the Subdivision Basidiomycotina, Order Ustilaginales, and to
their use in the control of pests, particularly pestiferous mites
(Acari) and pathogenic plant fungi, especially powdery mildews.
[0010] As presently claimed, the present invention relates to a
substantially pure culture of the fungus Meira geulakonigae nom.
prov. (M. geulakonigae), a substantially pure culture of the fungus
Meira argovae nom. prov. (M. argovae), and a substantially pure
culture of the fungus Acaromyces ingoldii nom. prov. (A. ingoldii),
as well as their respective conditioned media.
[0011] All of the fungi cultures of the invention are biologically
pure.
[0012] The three new fungi species have been deposited in the
Centraal Bureau voor Schimmelcultures (CBS), Institute of the Royal
Netherlands Academy of Arts and Science, under the provisions of
the Budapest Treaty for the Deposit of Microorganisms.
[0013] The present invention also relates to biologically active
products derived from fungi selected from the group consisting of
M. geulakonigae, M. argovae and A. ingoldii, wherein said
biological activity is miticidal and/or fungicidal.
[0014] The present invention provides a composition for the control
of mites and a composition for the control of fungi. Both
compositions comprise a biocontrol agent derived from any or at
least one of the fungi of the invention, and/or from a product
thereof. The biocontrol agent consists of spores and/or products
thereof. The miticide and the fungicide composition may further
comprise an agriculturally compatible carrier, extracted from a
liquid culture of the respective fungi. In this respect, said
carrier may be selected from the group consisting of water, culture
medium, oils or any other adjuvants or carriers.
[0015] The miticide composition of the invention is available in
the form of a liquid suspension containing about between 10.sup.7
to 10.sup.10 spores/ml, preferably about 10.sup.9 spores/ml.
[0016] The fungicide composition of the invention is available in
the form of liquid suspension containing between about 10.sup.7 to
about 10.sup.10 spores/ml, preferably about 10.sup.8 spores/ml.
[0017] Both miticide and fungicide compositions of the invention
may further comprise a conventional adjuvant used in formulations
for biocontrol agents.
[0018] It is another aspect of the present invention to provide a
method for producing a fungicide and/or a miticide, wherein the
active ingredient of said fungicide/miticide is any one of M.
geulakonigae, M. argovae, or A. ingoldii, or any combination
thereof, and/or a culture thereof, and/or any product or derivative
thereof. This process comprises culturing a fungus of the
invention, and optionally isolating said active ingredient from the
culture.
[0019] In a particular embodiment, the method comprises the steps
of: (a) cultivating, under aerobic or anaerobic conditions, any one
of the fungi of the invention in an appropriate culture medium, (b)
harvesting the culture; (c) separating the spores from the liquid
medium; and either (d) suspending the spores in an agriculturally
compatible liquid carrier to give a fungicide/miticide in liquid
form; or (e) lyophilizing the spores, to give a dry form
fungicide/miticide. Said dry form fungicide/miticide can be further
dissolved in an agriculturally compatible liquid carrier. In this
method, the fungus is to be cultured at a temperature of from about
20.degree. C. to about 30.degree. C., preferably from about
23.degree. C. to about 27.degree. C., and more preferably at about
25.degree. C. The pH of the fungi culture should be from about 5.1
to about 5.6, preferably a pH of 5.1 is ideal.
[0020] Lastly, the invention provides a method for controlling mite
and/or fungi infestations in plants susceptible thereto, which
comprises applying to the plant the miticide or the fungicide
composition of the invention. The mite infestation may be caused by
Phyllocoptruta oleivora (citrus rust mite) or other rust and gall
mites, by Tetranychus cinnabarinus (carmine spider mite), and/or
Panonychus citri (citrus red mite) or other spider mites, while the
fungal infestation is usually due to Sphaerotheca fusca (powdery
mildew) or other powdery mildews. Any commercial plants susceptible
to spider mites, rust mites, gall mites or to phytopathogenic
fungal diseases, in particular to powdery mildew, may be treated
with the miticide and/or with the fungicide composition of the
invention.
[0021] All the above and other characteristics and advantages of
the invention will be further understood through the following
illustrative and non-limitative description of preferred
embodiments thereof, with reference to the appended figures.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1: Dendrogram showing the taxonomic classification of
the three fungal species.
[0023] AS001 represents Acaromyces ingoldii nom. prov.; AS004
represents Meira geulakonigae nom. prov.; and AS005 represents
Meira argovae nom. prov.
[0024] FIG. 2: Photographs of the three new fungal species.
[0025] FIG. 3: Effect of treatment by Meira geulakonigae nom. prov.
(M. geulakonigae) on the extent of damage caused by powdery mildew
(Sphaerotheca fusca) to infected cucumber plants.
[0026] Graph showing the coverage of cucumber leaves (in
percentage) by powdery mildew (PM) before or after infection, and
treated or untreated with M. geulakonigae. Different treatments are
represented as: W, no infection, water only; Mg, no infection, M.
geulakonigae only; PM-W, infection first, then water; PM-Mg,
infection first, then M. geulakonigae; W-PM, water first, then
infection; Mg-PM, M. geulakonigae first, then infection. A, B, C
and D denote 13, 20, 27 and 53 days post-treatment, respectively.
Abbreviations: L. cov., leaf coverage.
[0027] FIG. 4: Meira geulakonigae nom. prov. increases the yield of
cucumber plants infected by powdery mildew (Sphaerotheca
fusca).
[0028] Graph showing the average cucumber yield in weight (g) of
plants infected with powdery mildew, before or after treatment with
a suspension of M. geulakonigae spores. Different treatments are
represented by the abbreviations as in FIG. 3. Abbreviations: Wt.,
weight.
[0029] FIG. 5A-B: Meira geulakonigae nom. prov. reduces foliage
mortality and increases the yield of cucumber plants.
[0030] FIG. 5A: Graph showing the effect of M. geulakonigae on (A)
number of cucumber leaves killed by powdery mildew (Sphaerotheca
fusca).
[0031] FIG. 5B: Graph showing the effect of M. geulakonigae on
cucumber yield. Different treatments are represented by the
abbreviations as in FIG. 3. Abbreviations: No. L., number of
leaves; Wt., weight.
[0032] FIG. 6A-B: Meira geulakonigae nom. prov. reduces the
covering of cucumber plants by powdery mildew (Sphaerotheca
fusca).
[0033] FIG. 6A: General view of the effect of M. geulakonigae
treatment on cucumber plants infected by powdery mildew (PM) in a
pilot commercial greenhouse. Right: natural infection of PM on
cucumber plants that were treated with M. geulakonigae. Left:
natural infection of PM on cucumber plants.
[0034] FIG. 6B: Close-up of (A). Right: natural infection of PM on
cucumber plants. Left: natural infection of PM on cucumber plants
that were treated with M. geulakonigae.
[0035] Abbreviations: Mg, M. geulakonigae; n.-tr., non-treated;
tr., treated.
[0036] FIG. 7: Meira geulakonigae nom. prov. reduces carmine spider
mite (Tetranychus cinnabarinus) infestation on cucumber plants.
[0037] Graph showing the number of leaves infested by at least one
carmine spider mite on cucumber leaves treated with M.
geulakonigae. Data collected from three examinations over a period
of 16 days, in a pilot commercial greenhouse. Different treatments
are represented by the abbreviations as in FIG. 3. Abbreviations:
No. L., number of leaves.
[0038] FIG. 8: Meira geulakonigae nom. prov. increases the
mortality of citrus rust mite (Phyllocoptruta oleivora) on citrus
leaves.
[0039] Graph showing the percent mortality of citrus rust mite on
citrus leaves, after treatment with M. geulakonigae (10.sup.9
spores/ml).
[0040] Abbreviations: T, treated; C, control; Mort., mortality.
DETAILED DESCRIPTION OF THE INVENTION
[0041] The subject of the present invention concerns the use of
biocontrol agents of fungal origin to control certain pests.
Specifically exemplified herein is the use of the novel species of
the invention, Meira geulakonigae nom. prov. (M. geulakonigae),
Meira argovae nom. prov. (M. argovae) and Acaromyces ingoldii nom.
prov. (A. ingoldii).
[0042] Note 1: The finding of the species-subject of this patent
application was not published at the time of submitting this
application. Therefore, for the purposes of the art of Taxonomy the
names of the respective species are considered provisional, and
require the notation "nom. prov.".
[0043] Note 2: For the purposes of this patent application, Meira
geulakonigae nom. prov. is equivalent to Meira geulakonigii nom.
prov.
[0044] A further aspect of the subject invention includes
compositions and formulations which are highly effective in
delivering the biocontrol agent to the target pest and are
apparently devoid of any adverse effects on the treated plants
and/or the environment.
[0045] Substantially pure cultures of A. ingoldii, M. geulakonigae
and M. argovae (all nom. prov.) have been deposited in the Centraal
Bureau voor Schimmelcultures (CBS), Institute of the Royal
Netherlands Academy of Arts and Science (P.O. Box 85167, 3508 AD,
Utrecht, The Netherlands), as follows:
TABLE-US-00001 Patent Deposit Culture restricted No. Date Accession
No. Acaromyces ingoldii CBS 109901 13 Dec. CBS 110050 nom. prov.
L1/AS001 2001 Meira geulakonigae CBS 109902 13 Dec. CBS 110052 nom.
prov. L4/AS004 2001 Meira argovae CBS 109903 13 Dec. CBS 110053
nom. prov. L5/AS005 2001
[0046] The deposits are available as required by foreign patent
laws in countries wherein counterparts of the subject application,
or its progeny, are filed. However, it should be understood that
the availability of the deposits does not constitute a license to
practice the subject invention in derogation of patent rights
granted by a governmental action.
[0047] Further, the subject culture deposits were stored and made
available to the public in accord with the provisions of the
Budapest Treaty for the Deposit of Microorganisms, i.e.; they were
stored with all the care necessary to keep them viable and
uncontaminated for a period of at least five years after the most
recent request for the furnishing of a sample of a deposit, and in
any case, for a period of at least thirty (30) years after the date
of deposit or for the enforceable life of any patent which may
issue disclosing the cultures. The depositor acknowledges the duty
to replace the deposit(s) should the depository be unable to
furnish a sample when requested, due to the condition of a deposit.
All restrictions on the availability to the public of the subject
culture deposits will be irrevocably removed upon the granting of a
patent disclosing them.
[0048] It is to be understood, that a substantially pure culture is
a culture that may comprise variations which a person of ordinary
skill in the art would know would not defeat the stated purpose of
the invention, and which would still be within the present
inventive concept.
[0049] Preferred cultures, according to the invention, are
biologically pure cultures. By "biologically pure", as used herein,
is meant a culture free of deleterious viable contaminating
microorganisms.
[0050] Two of new fungal species of the invention, namely M.
geulakonigae, and A. ingoldii (all nom. prov.), were obtained from
cadavers of the citrus rust mite, found on citrus leaves in two
ecosystems in Israel, specifically the Upper Galilee region and the
south of the Coastal Plain. The third new fungal species, M.
argovae (nom. prov.), was isolated from the cadaver of the carmine
spider mite found on the leaf of a castor bean (Ricinus communis)
bush, in Ness Ziona, in the central Coastal Plain of Israel.
[0051] Taxonomic, morphological and molecular characterizations
were made using the methods recommended by the CBS. It is to be
mentioned that due to their minute size, all of the novel species
could only be characterized by molecular biology techniques. The
three species are within the Subdivision Basidiomycotina, Order
Ustiginales. The dendrogram shown (FIG. 1) is a representation of
the outcome of a phylogenetic analysis of a molecular study for the
correct placement of the fungi, indicating the clear separation of
the three new species. Along the dendrogram, the three new species
are denoted as: Acaromyces ingoldii nom. prov., AS001; Meira
geulakonigae nom: prov., AS004; and Meira argovae nom. prov.,
AS005. More precise determinations were done with the Internal
Transcribed Spacer of Large Sub Units (ITS-LSU), and run on the
Unweighted Pair-Group Method using Arithmetic Averages program
(UPGMA), and the LSU-ITS Principal Component Analysis (PCA)
methods. The resulting drawings demonstrate the unambiguous
positioning of the three new species (FIG. 1 and data not
shown).
[0052] Photographs of the three fungi species are presented in FIG.
2. The three species were grown on potato dextrose agar (PDA) for
11 days at about 25.degree. C. FIG. 2a shows A. ingoldii, whose
colony is whitish, whereas M. geulakonigae (FIG. 2b) and M. argovae
(FIG. 2c) are brownish. Both M. geulakonigae and M. argovae
colonies secrete pigments into the media, and M. argovae secrets
higher quantities. The three species demonstrated differences in
colony growth pattern. M. geulakonigae colony is more woolish,
whereas M. argovae shows an almost segmented growth pattern. Fungal
growth was similar under light and dark conditions. None of the
three species has shown any chitinolytic or proteolytic
activities.
[0053] Thus, in a first aspect, the invention provides
substantially pure cultures of each of the fungi M. geulakonigae,
M. argovae and A. ingoldii (all nom. prov.). Specifically, the
invention provides substantially pure cultures of the fungi
deposited at the CBS under Accession Nos. CBS 110052, CBS 110053
and CBS 110050.
[0054] It is known in the art how to obtain mutants of
microorganisms without altering the characteristics thereof. For
instance, mutants may be obtained by treatment with physical or
chemical mutagens, such as UV light, X-rays, gamma-rays and
chemicals such as n-methyl-N'-nitro-N-nitrosoguanidine. It is also
known in the art how to obtain natural variants, e.g. by screening
cultures of the parental strain. Therefore, the invention also
pertains to the mutants or variants of M. geulakonigae, M. argovae
and A. ingoldii (all nom. prov.) which retain the characteristics
described herein, particularly mutants of the fungi deposited at
the CBS under Accession Nos. CBS 110052, CBS 110053 and CBS
110050.
[0055] In one embodiment, the fungi of the invention may be
cultured by any methods known in the art for their culture, e.g.
anaerobic cultivation, aerobic cultivation on agars, or submerged
aerobic fermentation in fermentors. Preferably, the culture is
fermented submerged.
[0056] The media for growth or fermentation may contain
assimilatable carbon sources and digestible nitrogen sources.
Suitable carbon sources include, but are not limited to, glucose,
lactose, mannitol, dextrin, corn, starch, xylose, fructose, lactose
and the like.
[0057] Suitable nitrogen sources include, but are not limited to,
natural nitrogen-containing materials and the products thereof,
e.g. meat extracts, peptones, corn infiltration solutions, yeast
extracts, soy bean meals, tryptones, cotton seed meals, wheat
meals, and the like. Organic or inorganic nitrogen-containing
materials may also be used, e.g. urea, nitrates and ammonium salts,
such as sodium nitrate, ammonium acetate, ammonium chloride,
ammonium sulfate, ammonium phosphate, and the like.
[0058] Optionally, the media for cultivating M. geulakonigae, M.
argovae and A. ingoldii (all nom. prov.) may also contain inorganic
salts, trace elements, and growth stimulators.
[0059] The inorganic salts suitable for use in the media for
cultivating M. geulakonigae, M. argovae and A. ingoldii (all nom.
prov.) include, but are not limited to, salts capable of yielding
zinc, sodium, magnesium, calcium, ammonium, chloride, carbonate,
sulfate nitrate, and the like.
[0060] In addition, essential trace elements necessary for the
growth and development of the organisms may also be included in the
culture media. Such trace elements commonly occur as impurities in
other components of the media in an amount sufficient to meet the
growth requirements of the organism.
[0061] The growth stimulators suitable for use in the invention
include, but are not limited to, glycerol and sodium salts, such as
sodium acetate, sodium glutamate, sodium tartarate, and the
like.
[0062] The fungi of the invention should be grown at temperatures
of from about 20.degree. C. to about 30.degree. C., preferably from
about 23.degree. C. to about 27.degree. C., more preferably at
about 25.degree. C.
[0063] As is customary in aerobic culture processes, sterile air is
blown into the vessel from the bottom while the medium is stirred
with conventional turbine impellers. In general, the aeration rate
and agitation rate should be sufficient to maintain a level of
dissolved oxygen of at least 30% of air saturation with an internal
vessel pressure of 0.2 bars.
[0064] The pH value in the fermentator varies along with the
culture media and the quantity of inocula used. Typically, the pH
value is maintained weakly acidic. If necessary, prior to
inoculation or during the fermentation, the pH value in the
fermentator can be appropriately adjusted to be weakly acidic e.g.
about pH 5.1.+-.0.2 to about pH 5.6. Preferably, the pH value prior
to inoculation is adjusted to about pH 5.1.
[0065] Any basic materials known in the art can be used to adjust
the pH value, e.g. alkaline metal salts, such as sodium salts, and
the like.
[0066] In principle, the time for cultivating M. geulakonigae, M.
argovae or A. ingoldii (all nom. prov.) should be enough to produce
a sufficient amount of spores. In general, the cultivation is
conducted for about 5 to about 8 days at the said temperatures,
preferably at 25.degree. C.
[0067] Furthermore, anti-foam agents can be appropriately added
during the fermentation to inhibit excessive foam formation. For
instance, polypropylene glycol of a molecular weight of about 2000
can be added to the culture medium at an amount of about 0.2
g/liter of culture medium.
[0068] The term "conditioned medium" used in the specification
refers to the whole contents in a flask or fermentor, including
both the cellular and non-cellular components, resulting directly
from the fermentation of any or a combination of the fungi of the
invention in an appropriate culture medium without being subject to
filtration, separation or purification.
[0069] Thus, the conditioned medium of any one of the fungi of the
invention is obtained upon cultivating the said fungi in the
above-specified culture medium and under the above-specified pH,
temperature and duration.
[0070] Example 1 shows the activity of M. geulakonigae, M. argovae
and A. ingoldii in the control of powdery mildew in the cucumber
plant, while in Examples 2 and 3, the three new fungi species are
used in the control of pest mites. Similarly, the conditioned
medium of the fungi of the invention shall be active in the control
of pest mites, such as the carmine spider mite (Tetranychus
cinnabarinus) the citrus red mite (Panonychus citri), the oriental
red mite, (Eutetranychus orientalis) and the citrus rust mite
(Phyllocoptruta oleivora), and phytopathogenic fungi. The active
ingredient in the conditioned medium is any compound that is toxic
and/or incompatible with the life or growth of pest mites and/or
phytopathogenic fungi.
[0071] The conditioned medium can be used directly for the control
of pest mites and phytopathogenic fungi or as part of a composition
of the invention, as described below.
[0072] In another aspect, the invention relates to a composition or
formulation for the control of pests, comprising as effective
ingredient any one of the fungi M. geulakonigae, M. argovae or A.
ingoldii (all nom. prov.), or any effective biocontrol agent
derived from said fungi, or progeny or mutants thereof or from a
culture medium used for growing the same, any product or any
mixture thereof.
[0073] In a preferred embodiment, the biocontrol agent is derived
from the group consisting of spores and conidia, preferably spores
and/or products thereof.
[0074] Any one of the fungi of the invention, M. geulakonigae, M.
argovae or A. ingoldii (all nom. prov.), may produce or contain a
compound, which may be a nucleotide, a protein, traces of metals,
that are, directly or indirectly, toxic or incompatible with the
life of pest mites and/or phytopathogenic fungi. These are herein
referred as products thereof, or more specifically biologically
active products thereof. Similarly, the products may also be
derived from the group consisting of spores and conidia, preferably
spores, derived from the fungi of the invention.
[0075] In Examples 2 and 3, mite-infested cucumber and citrus
seedlings plants are treated with M. geulakonigae and the level of
infestation significantly decreases when compared with
water-treated or untreated infested plants. Thus, in one
embodiment, the composition of the subject invention is intended
for the control of plant-infecting mites (Acari). In a preferred
embodiment, the pest mites are the carmine spider mite (Tetranychus
cinnabarinus) the citrus red mite (Panonychus citri), the oriental
red mite, (Eutetranychus orientalis) and the citrus rust mite
(Phyllocoptruta oleivora).
[0076] The composition for the control of mites comprises an
aqueous suspension, containing between about 10.sup.7 to 10.sup.10
spores/ml, preferably 10.sup.9 spores/ml. The mite control
composition of the invention may further comprise other active or
non-active agriculturally acceptable materials. These may be other
miticides and/or agriculturally acceptable carriers, diluents,
adjuvants and excipients.
[0077] The fungi and compositions of the present invention, and
their products may be used for the treatment and prevention of mite
infestation in plants that include, but are not limited to, all
varieties of citrus plants, apples, grapes, cucumbers, squash and
any other commercial plants that are attacked by phytophagous
mites.
[0078] In Example 1, M. geulakonigae is successfully used in the
treatment of powdery mildew-infested cucumber plants. Thus, in
another embodiment, the fungi and compositions of the present
invention and their products may be used for the control of
phytopathogenic fungi. In a preferred embodiment, the invention is
for the treatment and/or prevention of cucumber powdery mildew,
Sphaerotheca fusca infestation, as well as that of other powdery
mildews in different crops, and other genera and species of
phytopathogenic fungi.
[0079] Compositions for the control of fungi comprise an aqueous
suspension, containing between about 10.sup.7 to 10.sup.10
spores/ml, preferably 10.sup.9 spores/ml. The fungus control
compositions of the invention may further comprise other active or
non-active agriculturally acceptable materials. These may be other
miticides, fungicides, and/or agriculturally acceptable carriers,
diluents, adjuvants and excipients.
[0080] The fungi and anti-fungal compositions of the invention may
be used for the treatment of various crops. Such crops may be, but
are not limited to, cucumber, tomato, eggplant, pepper, apple,
grapes and all commercial crops susceptible to phytopathogenic
fungi.
[0081] In a further aspect, the invention comprises a method for
the production or preparation of a fungicide and/or a miticide,
wherein the active ingredient of said fungicide/miticide is any one
of M. geulakonigae, M. argovae, or A. ingoldii, or any combination
thereof, and/or a culture thereof, and/or any product or derivative
thereof. This process comprises culturing a fungus of the
invention, and optionally isolating said active ingredient from the
culture.
[0082] In a particular embodiment, the method comprises the steps
of: (a) cultivating, under aerobic or anaerobic conditions, any one
of the fungi of the invention in an appropriate culture medium; (b)
harvesting the culture; (c) separating the spores from the liquid
medium; and either (d) suspending the spores in an agriculturally
compatible liquid carrier to give a fungicide/miticide in liquid
form; or (e) lyophilizing the spores, to give a dry form
fungicide/miticide. Said dry form fungicide/miticide can be further
dissolved in an agriculturally compatible liquid carrier.
Optionally, the spores may be washed with deionized water after
they are separated from the liquid medium.
[0083] In the above-described method, the fungus is to be cultured
at a temperature of from about 20.degree. C. to about 30.degree.
C., preferably from about 23.degree. C. to about 27.degree. C., and
more preferably at about 25.degree. C. The pH of the fungi culture
should be from about 5.1 to about 5.6, preferably a pH of 5.1 is
ideal.
[0084] In one embodiment, the fungicide/miticide is an aqueous
suspension of spores, obtained through cultivating, under aerobic
or anaerobic conditions, any one of the fungi M. geulakonigae, M.
argovae or A. ingoldii (all nom. prov.) in an appropriate culture
medium.
[0085] In a second embodiment, the fungicide/miticide of the
invention is a lyophilized of fungus spores, obtained through the
above-described method, wherein the spores are from any one of the
fungi M. geulakonigae, M. argovae or A. ingoldii. This lyophilized
form may be advantageous for storage and handling of the
fungicide/miticide.
[0086] The composition may be prepared in any known manner, e.g. by
supplementing the active ingredient with agriculturally acceptable
carriers, auxiliaries or diluents, such as solvents, emulsifiers
and dispersants or surfactants.
[0087] Solvents suitable for use in the invention and its various
products include, but are not limited to, aromatics, e.g. xylene;
chlorinated aromatics, e.g. chlorobenzenes; paraffins, e.g. mineral
oil fractions; alcohols, e.g. methanol and butanol; ketones, e.g.
cyclohexanone; amines, e.g. ethanolamine and dimethylformamide; and
water, preferably deionized. When water is used, other organic
solvents may also be used as co-solvents.
[0088] Carriers suitable for use in the invention and its products
include, but are not limited to, ground natural or synthetic
minerals, e.g. kaolins, clays, talc, chalk, silica, silicates, and
the like.
[0089] Emulsifiers suitable for use in the compositions of the
invention include, but are not limited to, nonionic and anionic
emulsifiers, e.g. polyoxyethylene fatty alcohol ethers,
alkylsulfonates, arylsulfonates, and the like.
[0090] Dispersants suitable for use in the compositions of the
invention include, but are not limited to, lignosulfite waste
liquors and methylcellulose; and the like.
[0091] Suitable surfactants include, but are not limited to,
ligno-phenol-, naphthalene- and dibutylnaphthalenesulfonic acid,
fatty acids, alkyl- and alkylarylsulfonates, alkyl lauryl ether and
fatty alcohol sulfates, salts of sulfated hexa-, hepta- and
octadecanols and of fatty alcohol glycol ethers, condensates of
sulfonated naphthalene and its derivatives with formaldehyde,
condensates of naphthalene, or of the naphthalenesulfonic acids,
with phenol and formaldehyde, polyoxyethylene octylphenol ether,
ethoxylated iso-octyl-, octyl- or nonylphenol, alkylphenol
polyglycol ethers, tributylphenyl polyglycol ether, alkylauryl
polyether alcohols, isotridecyl alcohol, fatty alcohol/ethylene
oxide condensates, ethoxylated castor oil, polyoxyethylene alkyl
ethers or polyoxypropylene alkyl ethers, lauryl alcohol polyglycol
ether acetate, sorbitol esters, lignosulfite waste liquors or
methylcellulose.
[0092] In yet another aspect the invention comprises the use of the
novel species and their products in a method for the control and/or
prevention of mites (Acari) and/or fungi infestation in plants.
[0093] In one embodiment, the method comprises spraying the plant,
before or after an infestation by Acari or an infection by fungi,
with a composition comprising a suspension of spores of any one of
the fungi M. geulakonigae, M. argovae or A. ingoldii (all nom.
prov.), or a mixture of at least two thereof.
[0094] The plants grow very rapidly, and fungal infections and
acarine infestations happen repeatedly, also due to their
wind-borne nature. Thus, preferably, in the above-mentioned method,
the composition is to be applied on the plant weekly or bi-weekly,
during the season. This procedure is similar to that used with
chemical pesticides.
[0095] For the purpose of application, the miticide or fungicide
suspension can be used directly or formulated as compositions
suitable for spraying, atomizing, dusting, spreading or pouring.
For instance, the compositions can be formulated as ready-to-spray,
solutions, powders, suspensions, highly concentrated aqueous, oily
or other suspensions or dispersions, emulsions, oil dispersions,
pastes, dusts, or granules.
[0096] The non-toxic aqueous pesticide may also contain various
additives such as antioxidants, preservatives, pH neutralizers
and/or clarifiers.
[0097] In use, the non-toxic aqueous pesticide is diluted and
sprayed or misted on the infested host, whether plant or animal, to
directly contact the surface of the target pests. In some cases,
repeated applications may be required.
[0098] To enhance the efficiency of the application, the
fungicidal/acaricidal composition of the invention may also
comprise other active ingredients, such as herbicides,
insecticides, growth stimulators, fertilizers and the like.
[0099] Disclosed and described, it is to be understood that this
invention is not limited to the particular examples, process steps,
and materials disclosed herein as such process steps and materials
may vary somewhat. It is also to be understood that the terminology
used herein is used for the purpose of describing particular
embodiments only and not intended to be limiting since the scope of
the present invention will be limited only by the appended claims
and equivalents thereof.
[0100] It must be noted that, as used in this specification and the
appended claims, the singular forms "a", "an" and "the" include
plural referents unless the content clearly dictates otherwise.
[0101] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integers or steps.
[0102] The following examples are representative of techniques
employed by the inventors in carrying out aspects of the present
invention. It should be appreciated that while these techniques are
exemplary of preferred embodiments for the practice of the
invention, those of skill in the art, in light of the present
disclosure, will recognize that numerous modifications can be made
without departing from the spirit and intended scope of the
invention.
EXAMPLES
Experimental Procedures
Source of the Fungi:
[0103] Two of the fungal species, namely M. geulakonigae and A.
ingoldii (all nom. prov.), were obtained from cadavers of the
citrus rust mite in different sites in Israel, from the Upper
Galilee to the south of the Coastal Plain. The third species, M.
argovae (nom. prov.), was isolated from the carmine spider mite
found on a leaf of castor bean at the central Coastal Plain.
Large-Scale Production of Fungi:
[0104] All three fungi were mass-cultured in the laboratory on the
common solid medium, PDA (potato dextrose agar, Difco) and on the
liquid medium PB (potato broth, Difco), at the optimal temperature
of 25.degree. C. The fungi have a very rapid life cycle, and
maximal production of spores took place within 4 to 5 days. Thus,
very large amounts of spores were obtained within a short period of
time.
Preparation of the Suspension:
[0105] Spores were washed off from the culture grown on PDA with
deionized water and their amount (per ml) adjusted to the required
concentration with a haemocytometer. The spores were separated from
the liquid medium by centrifugation. Deionized water was added and
their concentration was adjusted as above.
Application of the Fungicide:
[0106] Experiments with powdery mildew (PM) were conducted in the
laboratory and in the greenhouse, and one experiment was conducted
in a pilot commercial greenhouse. Suspensions of M. geulakonigae
(2.times.10.sup.8 spores/ml) were sprayed onto cucumber leaves.
Leaf coverage by PM and number of leaves killed by the disease were
assayed, and the dry matter of the entire plants, and their yields,
were weighed.
Application of the Acaricide:
[0107] Plants were sprayed as described above, with a suspension of
M. geulakonigae of about 10.sup.9 spores/ml.
Example 1
[0108] Initial experiments were undertaken to assay the effect of
the three new fungi species M. geulakonigae, M. argovae and A.
ingoldii (all nom. prov.) on cucumber powdery mildew infecting
detached leaves. Treated leaves were sprayed with each one of the
fungi, while control leaves were not sprayed, and then all the
leaves were inoculated with powdery mildew. Disease was assessed as
the leaf area covered by the powdery mildew, and the experiment was
followed weekly for a period of four weeks. At the end of four
weeks, the results obtained were as follows. Control leaves had
between 50 to 100% of their area covered by the fungi within the
first week, which was then followed by leaf mortality. In contrast,
the leaves treated with either M. geulakonigae, M. argovae or A.
ingoldii (all nom. prov.) usually had less than 10% of their area
covered by the powdery mildew after 15 days. The infection covered
area then increased to about 25% at the end of four weeks, by which
time most leaves were still alive.
[0109] After the successful results obtained in the initial
experiments with all three fungi, a more systematic study was
performed using only M. geulakonigae for the treatment of powdery
mildew infection on cucumbers. Thus, cucumber plants were sprayed
with a M. geulakonigae (Mg) suspension, before or after infection
by powdery mildew (PM). Various treatment protocols were used: no
infection and only water treatment (W in FIG. 3); no infection and
only M. geulakonigae treatment (Mg in FIG. 3); PM infection first,
and then water treatment (PM-W in FIG. 3); infection first, and
then M. geulakonigae treatment (PM-Mg in FIG. 3); water treatment
first, and then infection with PM (W-PM in FIG. 3); M. geulakonigae
treatment first, then PM infection (Mg-PM in FIG. 3). The following
parameters were analyzed:
[0110] (a) The extent (percentage) of PM coverage on cucumber
leaves was evaluated according to the method of Horsfall &
Barratt [Horsfall, J. G. & Barratt, R. W. Phytopathology. 35:
655 (1945)] 13, 20, 27 and 53 days post-treatment, and compared to
control plants treated with water. PM coverage was faster in the
absence of M. geulakonigae treatment (FIG. 1, W, PM-W and W-PM)
than in its presence (Mg, PM-Mg and Mg-PM), showing the significant
bio-control effect of M. geulakonigae on the pathogenic fungus.
[0111] (b) The yield of cucumbers (in grams, g) from plants sprayed
with M. geulakonigae was consistently and significantly higher than
the yield of cucumbers from untreated plants (FIG. 4).
[0112] (c) The number of dead leaves on cucumber plants sprayed
with a suspension of Mg was significantly lower than on plants
infected by PM but not given the Mg treatment (FIG. 5A).
[0113] (d) The dry weight of M. geulakonigae treated plants was
significantly higher than the dry weight of untreated plants (FIG.
5B).
[0114] Clear evidence of the bio-control effect of M. geulakonigae
on powdery mildew infecting cucumbers can be appreciated in the
photographs presented in FIG. 6.
Example 2
[0115] The effect of M. geulakonigae treatment on the plant-feeding
carmine spider mite (Tetranychus cinnabarinus) infesting cucumbers
was tested. This mite is a major pest of many crops. The plants
were treated using the same protocol as described in Example 1. In
order to avoid destructive sampling, the number of leaves infested
by at least one live adult spider mite was counted. The data was
obtained from 5 leaves per plant and 6 plants per treatment. Thus,
a total of 30 leaves were evaluated on each observation date. All
leaves that were not treated with M. geulakonigae had significantly
larger spider mite populations (FIG. 7) and at times showed heavy
mite damage.
Example 3
[0116] One initial experiment was conducted with the major citrus
pest, the citrus rust mite Phyllocoptruta oleivora as target, using
all three new fungi for pest control. Table 1 shows the results
obtained for this initial experiment, the numbers represent the
percentage of mites killed after treatment, calculated with
Abbott's correction. The names of all three fungi are
provisional.
TABLE-US-00002 TABLE 1 Control A. ingoldii M. geulakonigae M.
argovae 15.2 96.5 95.3 92.3
[0117] Following the results of the initial experiment using the
three new fungi species, a second experiment was conducted on the
effect of M. geulakonigae on the citrus rust mite. Heavily-infested
citrus seedlings were sprayed with M. geulakonigae suspensions of
10.sup.9 spores/ml, obtained after liquid fermentation of the
fungus. Pest mortality was almost 100% (after Abbott's correction)
in the treated plants, as compared to about 30% in the untreated
control (FIG. 8).
* * * * *