U.S. patent application number 16/521326 was filed with the patent office on 2020-01-30 for lichen compounds that inhibit mycotoxin production.
The applicant listed for this patent is John E. Linz, Ludmila Roze, Frances Trail. Invention is credited to John E. Linz, Ludmila Roze, Frances Trail.
Application Number | 20200029575 16/521326 |
Document ID | / |
Family ID | 69179317 |
Filed Date | 2020-01-30 |
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United States Patent
Application |
20200029575 |
Kind Code |
A1 |
Trail; Frances ; et
al. |
January 30, 2020 |
LICHEN COMPOUNDS THAT INHIBIT MYCOTOXIN PRODUCTION
Abstract
Described herein are methods for obtaining useful compounds from
lichens.
Inventors: |
Trail; Frances; (Mason,
MI) ; Roze; Ludmila; (Okemos, MI) ; Linz; John
E.; (Lansing, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Trail; Frances
Roze; Ludmila
Linz; John E. |
Mason
Okemos
Lansing |
MI
MI
MI |
US
US
US |
|
|
Family ID: |
69179317 |
Appl. No.: |
16/521326 |
Filed: |
July 24, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62702424 |
Jul 24, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01N 65/00 20130101;
C12Q 1/18 20130101; A01N 65/03 20130101; A01N 63/30 20200101; A01N
63/20 20200101; A01N 63/30 20200101; A01N 25/02 20130101; A01N
63/20 20200101; A01N 65/03 20130101; A01N 63/20 20200101; A01N
25/02 20130101; A01N 65/03 20130101; A01N 25/02 20130101; A01N
65/00 20130101; A01N 25/02 20130101 |
International
Class: |
A01N 65/00 20060101
A01N065/00; C12Q 1/18 20060101 C12Q001/18 |
Claims
1. A method comprising (a) extracting a lichen sample with alcohol,
ethyl acetate, acetone or a combination thereof to provide a lichen
extract; (b) mixing the extract, a component of the extract, or a
compound obtained from the extract with Aspergillus parasiticus
cells or Fusarium graminearum cells to form an assay mixture; and
(c) measuring whether growth, sporulation, or mycotoxin production
by the Aspergillus parasiticus cells or Fusarium graminearum cells
is different from a control.
2. The method of claim 1, wherein the control is an average amount
of growth, sporulation, or mycotoxin production by Aspergillus
parasiticus cells or Fusarium graminearum cells that were not mixed
with the extract, the component of the extract, or the compound
obtained from the extract.
3. The method of claim 1, wherein the assay mixture comprises a
culture medium that promotes mycotoxin production by the
Aspergillus parasiticus cells or the Fusarium graminearum
cells.
4. The method of claim 1, further comprising isolating an extract
component or compound that reduces the growth, sporulation, or
mycotoxin production by the Aspergillus parasiticus cells.
5. The method of claim 1, further comprising isolating an extract
component or compound that reduces the growth, sporulation, or
mycotoxin production by the Fusarium graminearum cells.
6. The method of claim 1, wherein the cells are Aspergillus
parasiticus strain B62 cells.
7. The method of claim 1, further comprising isolating a component
or compound from the lichen extract.
8. The method of claim 7, further comprising mixing the component
of the extract, or a compound obtained from the extract with
Aspergillus parasiticus cells or Fusarium graminearum cells to form
an assay mixture; and measuring whether growth, sporulation, or
mycotoxin production by the Aspergillus parasiticus cells or
Fusarium graminearum cells is different from a control.
9. The method of claim 1, further comprising applying the lichen
extract, the component of the extract, or a compound obtained from
the extract to structure, to a surface, to an agricultural crop, to
a storage bin, to a storage facility, to an animal feed, to a plant
seed, to a nut, to a plant part, to a plant product, or to a
combination thereof.
10. The method of claim 1, wherein toxin content of a surface, an
agricultural crop, a storage bin, a storage facility, an animal
feed, a plant seed, a nut, a plant part, a plant product, or a
combination thereof is reduced by applying a composition of the
lichen extract, the component of the extract, or the compound
obtained from the extract to the surface, agricultural crop,
storage bin, storage facility, animal feed, plant seed, nut, plant
part, plant product, or the combination thereof.
11. The method of claim 10, wherein the mycotoxin content is
reduced by at least 50%.
12. An extract obtained by the method of claim 1 that reduces
mycotoxin content by at least 50%.
13. A composition comprising a lichen extract, a component of a
lichen extract, or a compound obtained from a lichen extract,
wherein the lichen extract is a lichen extracted by an alcohol,
ethyl acetate, acetone or a combination thereof.
Description
[0001] This application claims benefit of priority to the filing
date of U.S. Provisional Application Ser. No. 62/702,424, filed
Jul. 24, 2018, the contents of which are specifically incorporated
herein by reference in their entity.
BACKGROUND OF THE INVENTION
[0002] Fungi pose a greater threat to plant and animal biodiversity
than other types of pathogens (protists, viruses, bacteria,
helminths), and the threat of fungi to ourselves, our food
security, and our environment is increasing (Fischer et al., Nature
484:186-194 (2012)). Human diseases caused by fungi (mycoses) are
on the rise concomitantly with the increase in numbers of people
with immunosuppressive conditions (i.e. AIDS, organ transplants,
cancer treatments). Infections of immunocompromised humans by fungi
including Aspergillus fumigatus and Cryptococcus neoformans have
increased due to their ability, which is fairly unique among fungi,
to grow at body temperatures. Acquired drug resistance in medically
relevant microorganisms, including fungi and bacteria, is another
severe threat to human health. In addition, fungi and fungal-like
organisms are responsible for the destruction of about 125 million
tons of major crops such as rice, wheat, maize, potatoes, and
soybeans each year.
[0003] History also shows that fungi and fungal-like organisms can
dramatically affect human health and food security. For example,
the Irish Potato Famine, which was caused by the late blight mold,
resulted in mass starvation, and the death of 1 million people in
the 1840s. More recently, a new strain of wheat rust from Africa.
UG99 (Uganda 99), which causes up to 100% crop loss, is spreading
across wheat growing regions of the world. Mycotoxins pose a threat
to human health on nearly every continent, and climate change is
shifting their appearance to new areas (Magan et al., Plant
Pathology 60:150-163 (2011)).
[0004] Among mycotoxin contamination of crops, one of the most
serious is aflatoxin contamination of crops. Aflatoxin is thought
to be the most potent naturally occurring carcinogen known. An
aflatoxin contaminated diet has been directly linked with elevated
rates of liver cancer, decreased immunity, kwashiorkor, and growth
stunting.
[0005] In western countries, amounts of aflatoxin contaminants in
crops are regulated to below 10 parts per billion. However,
outbreaks of aflatoxicosis are common in underdeveloped countries,
mostly among poorly nourished rural populations whose staple food
is maize. Large losses of crops contaminated with aflatoxin are
common. Worldwide, Aspergillus species cause significant losses in
major crops. The annual economic impact of aflatoxin contamination
on corn and peanut agriculture in the United States is thought to
exceed $1 billion dollars.
[0006] A second major class of mycotoxins is the trichothecenes,
including T-2 toxin, nivalenol, deoxynivalenol, and
diacetoxyscirpenol, produced by the fungi Fusarium, Cephalosporium,
Myrothecium, Stachybotrys (the black mold), Trichoderma, and
others. In western countries, levels of deoxynivalenol are advised
to be less than 1 ppm for finished grain products for human
consumption. The trichothecenes are also damaging to the health of
pigs, cattle, poultry, and particularly swine. Due to climate
change and global warming in recent years, mycotoxigenic fungi are
altering their ranges, moving into areas in which they were not
previously found.
SUMMARY
[0007] Described herein are methods that include extracting lichens
and testing the extracts so obtained for their effects on fungal
sporulation, fungal hyphal growth and fungal secondary metabolite
production. Also described are anti-fungal compositions, as well as
methods for determining the microbial source and the structure of
the active compounds.
[0008] One method involves extracting a lichen sample with alcohol,
ethyl acetate, acetone or a combination thereof to provide a lichen
extract, and measuring whether the lichen extract inhibits the
growth, sporulation, or mycotoxin production of a fungus.
[0009] Another method involves (a) extracting a lichen sample with
alcohol, ethyl acetate, acetone or a combination thereof to provide
a lichen extract; (b) mixing the extract, a component of the
extract, or a compound obtained from the extract with Aspergillus
parasiticus strain B62 cells to form an assay mixture; and (c)
measuring whether growth, sporulation, or aflatoxin production by
the Aspergillus parasiticus strain B62 cells is different from a
control.
[0010] Another method involves extracting a lichen as in (a)
described above, and (b) mixing the extract, a component of the
extract, or a compound obtained from the extract with Fusarium
graminearum to form an assay mixture; and (c) measuring whether
growth or deoxynivalenol or 15-acetyl deoxynivalenol or nivalenol
production of variations thereof by the Fusarium graminearum cells
is different from a control.
[0011] The methods can be used to provide useful lichen extracts,
compounds, or components of the extracts.
[0012] Compositions are also described herein that can include a
carrier and an extract, compound, or a component of an extract
obtained, for example, by the methods described herein.
[0013] Such compositions, extracts, compounds, and extract
components are useful for inhibiting fungal growth, fungal
sporulation, toxin production, or a combination thereof. For
example, the compositions, extracts, compounds, and extract
components can reduce mycotoxin or toxin production by at least
10%, at least 20%, at least 30%, at least 40%, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%,
or at least 99%.
[0014] The extracts are stable, and retain the ability to inhibit
fungal growth, fungal sporulation, fungal toxin production, or a
combination thereof for at least ten years, or at least 20 years,
or at least 30 years.
[0015] The compositions, extracts, compounds, and extract
components can be applied to structures, surfaces, agricultural
crops, storage bins, storage facilities, animal feed, plant seeds,
nuts, plant parts, plant products, or a combination thereof. The
compositions, extracts, compounds, and extract components can be
applied to surfaces in bathrooms, kitchens, closets, basements,
attics, entryways, cabinets, boats, barns, animal shelters,
warehouses, food storage facilities, and other such surfaces. The
compositions, extracts, compounds, and extract components can be
applied to grain-producing plants, nut-producing plants,
vegetable-producing plants, fruit-producing plants,
starch-producing plants, fiber-producing plants, fodder-producing
plants, grains, nuts, vegetables, fruits, starch, fibers, flour,
fodder, leaves, stock, seeds, oil, or a combination thereof. The
compositions, extracts, compounds, and extract components can be
applied to almonds, barley, betel nuts, brazil nuts, cashews,
chestnuts, coconut, coffee, corn, flour, hazelnuts, macadamia nuts,
oats, pecans, peanuts, pine nuts, pistachios, rice, rye, sesame
seeds, soybean, spices, walnuts, wheat, or combinations
thereof.
[0016] Such methods can reduce the mycotoxin, fungal spore, or
fungal content in the surfaces, agricultural crops, storage bins,
storage facilities, animal feeds, plant seeds, nuts, plant parts,
plant products, or other places.
DESCRIPTION OF THE FIGURES
[0017] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0018] FIG. 1A-ID illustrate some types of lichens and a method for
assaying lichens to isolate bioactive lichen compounds with novel
activities. FIG. 1A shows an image of Cladonia spp. lichens that
grow on the soil. FIG. 1B shows an image of Physica spp. lichens
that attach to tree branches. FIG. 1C shows an image of Usnea spp.
lichens that attach to branches. FIG. 1D is a schematic diagram
illustrating methods for isolating bioactive lichen compounds with
novel activities.
[0019] FIG. 2A-2E illustrate high through-put assays in 24 well
plates for antifungal effects of lichen extracts on the fungus
Aspergillus parasiticus strain B62. The orange-red pigment is a
precursor and indicator of aflatoxin production. FIG. 2A shows a
3-day old culture of Aspergillus parasiticus strain B62 that was
not treated (control). FIG. 2B shows a 3-day old culture of
Aspergillus parasiticus strain B62 treated with an aflatoxin
biosynthesis inhibitor. FIG. 2C shows a 3-day old culture of
Aspergillus parasiticus strain B62 treated with a growth inhibitor.
FIG. 2D shows a 7-day old culture of Aspergillus parasiticus strain
B62 illustrating spore production (control). FIG. 2E shows a 7-day
old culture of Aspergillus parasiticus strain B62 treated with a
sporulation inhibitor. Plates with 24 wells were used for these
assays.
[0020] FIG. 3A-3C illustrate that extracts of Cladonia cristatella
inhibit secondary metabolite production by Aspergillus parasiticus
strain B62. FIG. 3A shows a culture of Cladonia cristatella
mycobiont generated from ascospores. FIG. 3B shows a top view of
Aspergillus parasiticus strain B62 cultures illustrating that
extracts of Cladonia cristatella mycobiont grown in liquid or solid
culture inhibit secondary metabolite production by Aspergillus
parasiticus strain B62. FIG. 3B shows a bottom view of Aspergillus
parasiticus strain B62 cultures illustrating that extracts of
Cladonia cristatella mycobiont grown in liquid or solid culture
inhibit secondary metabolite production by Aspergillus parasiticus
strain B62. Arrows indicate the orientation of the assay plate such
that the top and bottom of the same well is indicated.
[0021] FIG. 4A-4B illustrates that extracts of Evernia prunastri
strongly inhibit hyphal growth and secondary metabolite production
by Aspergillus parasiticus strain B62. FIG. 4A shows an image of
Evernia prunastri. FIG. 4B illustrates that ethanol extracts of
Evernia prunastri inhibit growth and aflatoxin accumulation in
Aspergillus B62 at 3 different concentrations.
[0022] FIG. 5A-5B illustrates of the stability of secondary
metabolites of lichen species Porpidia (P) and Lecidea (L) as shown
by thin layer chromatography (TLC). FIG. 5A shows TLC separation of
Porpidia (P) and Lecidea (L) extracts collected from an herbarium
specimen in 2006. FIG. 5B shows TLC separation of Porpidia (P) and
Lecidea (L) extracts collected from the same herbarium specimen in
2014. As shown, the secondary compounds in stored lichens were
stable.
[0023] FIG. 6 illustrates that extracts of two lichens (Evernia
pruastris [1], and Hypogymnia physodes [57]) whose extracts reduced
15-acetyl-deoxynivalenol (15-ADON) accumulation in the assay
described for FIG. 2 in comparison with cultures that were treated
with equivalent amounts of solvent (ethanol or acetone).
[0024] FIG. 7A-7B illustrate that lichen extracts inhibit
deoxynivalenol (DON) accumulation by Fusarium graminearum. FIG. 7A
shows that acetone extracts of lichens Evernia pruastris [1] and
Hypogymnia physodes [57] reduced DON production by cultured
Fusarium graminearum. FIG. 7B shows that acetone extracts of
lichens Cladonia sylvatica [15], Usnea strigosa [33], Cladonia
digitate [58], Ramalina fastigiate [62], Ramalina fraxinea [98],
Platismatia glauca [100], Evernia prunastri [112], and Cladonia
crispata [118] reduced DON production by cultured Fusarium
graminearum.
[0025] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
DETAILED DESCRIPTION
[0026] As described herein, lichens can produce a plethora of
unique secondary compounds, which are useful in medical,
agricultural and food safety applications. Lichens exhibit
significant longevity and robustness despite a close association
with diverse microbes and harsh environmental conditions. Lichens
provide model organisms for identifying and evaluating compounds
that can be useful as anti-fungal agents. As described herein,
extracts from lichen species were prepared, their activities were
tested, and useful compounds have been identified and isolated.
Lichens
[0027] Lichens are some of the longest-living organisms known.
Despite their slow growth, they very rarely appear die of disease.
The lichenized fungus establishes the main lichen thallus in
association with an alga or cyanobacterium, and has been recently
shown, a basidiomycete yeast is widely included (Spribille et al.
Science 3S3: 488-492 (2006)). This scaffold becomes a niche for a
variety of other filamentous ascomycetes and basidiomycetes,
yeasts, bacteria, and occasionally insects.
[0028] Lichens are defined by symbiotic interactions between a
fungus and photosynthetic partners, green alga or cyanobacterium.
Most species of fungi, including lichenized fungi, produce a
plethora of secondary metabolites, which are species-specific
nonessential metabolites that are highly diverse and have
biological activity. These compounds provide unique functions that
can aid organisms in niche-specific adaptations and can serve as
defense against disease and predation.
[0029] Antibiotics such as penicillin, and cephalosporin are
derived from fungi, and fungal derived antifungal griseofulvin is
used to treat athlete's foot and toe-nail fungus. Other unique
fungal metabolites used in medical applications include:
cyclosporins (immunosuppressants), statins (cholesterol lowering
drugs) and, fumagillin and pseurotin (angiogenesis inhibitors). One
major group of agricultural fungicides, the strobilurins, was
originally discovered in extracts of the fungus Strobilurus
tenacellus.
[0030] The methods described herein can provide new types of
compounds with useful activities. The inventors can use a unique
MSU resource, the Lichen Herbarium, to identify novel compounds to
develop into anti-fungal products for medicine and agriculture. The
lichen collection in the Plant Biology Department at MSU, an NSF
funded herbarium, houses more than 120,000 specimens and has the
greatest taxonomic and geographical diversity among its collection
compared to all other lichen herbaria in North America.
[0031] A variety of types of lichens can be used in the methods
described herein and can be a source of useful compounds including,
for example, crustose lichens, foliose lichens, leprose lichens,
squamulose lichens, and fructose lichens.
[0032] Lichens have an algal species associated with a fungal
species. Algal species associated with lichens are in the genera
Nostoc, Trebouxia and Trentephlia, which comprise the algae in 90%
of lichens. These algae can be found free living.
[0033] The variation in lichen morphology and physiology is
dictated by the fungus. It is therefore most likely that compounds
that are uniquely produced by a species of lichen are produced by
the fungus.
[0034] In some cases, the lichen can include species of the genus
Acarospora, Arctoparmelia, Amandinea, Aspicilia, Austroparmnelina,
Baeonmyces, Biatorina, Buellia, Bryoria, Byssoloma, Calicium,
Cladonia, Caloplaca, Candelariella, Cetraria, Cetrelia,
Chrysothrix, Cladia, Cladonia, Coenogonium, Collerna, Conotrema,
Cryptothecia, Dendrographa, Dermatocarpon, Dictyonema, Diploicia,
Diploschistes, Dirinaria, Endocarpon, Flavopanctelia,
Flavoparmelia, Flavopunctelia, Fulgensia, Fuscopannaria, Graphina,
Graphis, Graphis, Gyrophora, Haematomma, Heppia, Herpothallon,
Herpothalon, Heterodea, Heterodermia, Hypocenomyce, Hypogynmia,
Lasallia, Lecanora, Lecidea, Leioderma, Lepraria, Letharia,
Leptogiun, Lichen, Lecidea, Lichenomphalia, Lobaria, Menegazzia,
Metus, Multiclavula, Myelorrhiza, Neophyllis, Nephronma, Niebla,
Nostoc, Ochrolechia, Opegrapha, Opegrapha, Pannaria, Paraparmelia,
Paraporpidia, Parmelia, Parmeliella, Parmeliopsis, Parmotrema,
Peltigera, Peltigera, Peltula, Pertusaria, Pertusaria, Pertusaria,
Pertusaria, Pertusaria, Phaeographina, Phaeophyscia, Phlyctis,
Physcia, Physconia, Placidium, Placodium, Placopsis, Placopsis,
Platismatia, Pseudephebe, Pseudevernia, Pseudocyphellaria,
Pseudocyphellaria, Psora, Punctelia, Pyvxine, Ramalina, Rantboldia,
Relicina, Rhizocarpon, Rhizoplaca, Roccella, Sagedia, Siphula,
Sporopodium, Stereocaulon, Sticta, Stictis, Strigula, Teloschistes,
Tephromela, Tlutmnolia, Thysanothecium, Trapelia, Umbilicaria,
Usnea, Xanthomaculina, Xanthoparmelia, Xanthoria, or a combination
thereof.
[0035] Examples of lichens that can be used include those of the
type or species Acarospora citrina, Arctoparmelia centrifuga,
Amandinea punctate, Aspicilia fruticulosa, Aspicilia fruticulosa,
Aspicilia vagans, Aspicilia, Austroparmnnelina pruinata,
Austroparmelina pseudorelicina, Baeomvces heteromorphus, Biatorina
erysiboides, Bryoria fremontii, Buellia frigida, Buellia foecunda,
Buellia grimmniae, Buellia subcoronata, Byssoloma, Calicium
chrysocephalum, Caloplaca cinnabarina, Caloplaca cinnabarina,
Caloplaca, Candelariella aurella, Cetraria islandica, Cetrelia
olivetorum, Chrysothrix xanthine, Chrysothrix, Cladia aggregate,
Cladia moniliformis, Cladia retipora, Cladonia chlorophaceae,
Cladonia crispate, Cladonia crispata, Cladonia cristatela, Cladonia
didyma, Cladonia digitate, Cladonia fimbriate, Cladonia fimbriate,
Cladonia mitis, Cladonia pyxidate, Cladonia pyxidate, Cladonia
rangiferina, Cladonia stellaris, Cladonia sylvatica, Cladonia
ustulata, Coenogonium implexum, Collema coccophorum, Conotrema
urceolatum, Cryptothecia scripta, Dendrographa leucophaea,
Dernuatocarpon miniatum, Dictyonenm sericeum, Diploicia canescens,
Diploschistes thunbergianus, Dirinaria picta, Endocarpon pusillum,
Ephebe lanata, Evernia mesomorpha, Evernia prunastri,
Flavopanctelia flaventior, Flavoparmelia caperata, Flavoparmelia
rutidota, Flavopunctelia soredica, Fulgensia cranfieldii,
Fuscopannaria leucosticte, Fuscopannaria leucosticte, Fuscopannaria
subimmixta, Graphis bulacana, Graphina rubens, Graphis mnucronate,
Graphis treubii, Gyrophora murina, Haematomma africanum, Haematomma
eremaeum, Heppia despreauxii, Herpothalon rubrocinctum, Heterodea
beaugleholei, Heterodea muelleri, Heterodermia leucomelos,
Hypocenomyce australis, Hypogynmia physodes, Hypogymnia pulverate,
Lasallia papulose, Lasallia pustulata, Lecanora caesiorubella,
Lecanora circumborensis, Lecanora conizaeoides, Lecanora epibryon,
Lecanora pseudistera, Letharia vulpina, Letharia vulpine, Lecidea
lerrena, Leioderma pycnophorum, Lepraria lobfilcans, Lepraria
lohiticans, Leptogium saturninum, Lichen caninus, Lichen
ceuthocarpus, Lichen murorutm, Lichen rufescens, Lichen sylvaticus,
Lichenomphalia chromacea, Lecidea psora, Lobaria pulmonaria,
Menegazzia platytrema, Metus conglomeratus, Multiclavula mucida,
Myelorrhiza antrea, Neophyllis melacarpa, Nephroma arctica,
Nephroma cellulosum, Niebla cephalota, Nostoc, Ochrolechia
tartarea, Opegrapha varia, Opegrapha venosa, Parmelia sulcate,
Parmeliella plumbea, Pannaria sphinctrina, Paraparmelia
lithophiloides, Paraporpidia leptocarpa, Parmelia signifera,
Parmeliopsis chlorolecanorica, Parmotrenma perlatum, Peltigera
canina, Peltigera trumlata, Peltula euploca, Parmelia sulcate,
Pertusaria erebescens, Pertusaria pseudodactylina, Pertusaria
subventosa, Pertusaria melaleucoides, Pertusaria novaezelandiae,
Pertusaria, Phaeographina lamii, Phaeophyscia orbicularis,
Phlyctis, Physcia aipolia, Physcia millegrana, Physconia deters,
Physcia stellaris, Placidiumn squamulosum, Placodium murorum,
Placopsis perrugosa, Placopsis, Platismatia glauca, Pseudephebe
pubescens, Pseudevernia farinacea, Pseudevernia furfuracea,
Pseudocyphellaria berberina, Pseudocyphellaria billardierei,
Pseudocyphellaria crocata, Pseudocyphellaria freycinetii,
Pseudocyphellaria gilva, Pseudocyphellaria norvegica,
Pseudocyphellaria multifida, Pseudocyphellaria gilva,
Pseudocyphellaria, Psora crystallifera, Psora decipiens, Punctelia
borreri, Punctelia subrudecta, Pyxine cocoes, Ramalinafarinacea,
Ramalina fastigiate, Ramalina fastigiate, Ramalinafraxinea,
Ramalina menziesii, Ramalina siliquosa, Ranualina siliquosa,
Caloplaca sp., Tephromela atra, Ramboldia petraeoides, Relicina
gemmulosa, Rhizocarpon geographicum, Rhizocarpon geographicum,
Rhizoplaca melanophthalna, Roccella canariensis, Roccella
portentosa, Sagedia macrospora, Siphula coriacea, Sporopodium
vezdeanum, Stereocaulon ramulosum, Stereocaulon saxatile, Sticta
limbate, Stictis, Strigula smaragdula, Strigula subtilissima,
Teloschistes, Tephromela atra, Thamnolia vermicularis, Thamnolia
vermicularis, Thysanothecium scutellatum, Thysanothecium
sorediatum, Trapelia crystallifera, Trapelia, Umbilicaria
decussata, Umbilicaria hyperborean, Umbilicaria pustulata,
Umbilicaria hyperborean, Usnea antarctica, Usnea arizonica, Usnea
himantodes, Usnea inermis, Usnea rubicunda, Usnea scabrida, Usnea
strigosa, Usnea strigose, Usnea subfloridana, Usnea wasntathii,
Xanthomnaculina convoluta, Xanthoparmelia amplexula, Xanthoparmelia
arapilensis, Xanthoparmelia baeomycesica, Xanthopannelia
chlorochroa, Xanthopannelia convolute, Xanthoparmelia cravenii,
Xanthoparnmelia ewersii, Xanthoparmelia mougeotina, Xanthoparmelia
notlaa, Xanthoparmnelia praegnans, Xanthoparmelia
pseudoamphirantha, Xanthopannelia pulla, Xanthoparmelia reptans,
Xanthoparmelia semiviridis, Xanthoparmnnelia substrigosa,
Xanthoparmnelia taractica, Xanthopannelia versicolor, Xanthoria
filsonii, Xanthoria luhsseana, Xanthoria parietina, or a
combination thereof. For example, any of the lichens listed in
Appendix I of U.S. Provisional Application Ser. No. 62/702,424,
filed Jul. 24, 2018 can be used in the methods described herein and
can be a source of useful compounds.
[0036] In some cases, lichens such as Cladonia crispata, Cladonia
cristatella, Cladonia digitate, Cetraria islandica, Cetrelia
olivetorum, Cladonia sylwvaica, Evernia prunastri, Hypogymtnia
physodes, Platismatia glauca, Pseudevernia furfuracea, Rantalina
fastigiate, Ramnalina fraxinea, Usnea strigosa, or a combination
thereof can be extracted and/or can be a source of useful
compounds.
Mycotoxin
[0037] As indicated above, the most serious mycotoxin may be
aflatoxin in terms of contamination of crops. Aflatoxin is mainly
produced by strains of Aspergillus flavus and Aspergillus
parasiticus, which are ubiquitous in nature. Aspergillus flavus and
Aspergillus parasiticus have no specificity towards their hosts and
therefore can infect many different seeds of cereals, nut beans,
coffee beans and oil-rich seeds during cultivation, harvest and
post-harvest storage. These fungi infect crops such as maize and
peanut and produce aflatoxin under the tropical and subtropical
environments. Large amounts of aflatoxins can be produced,
especially under humid storage conditions.
[0038] Deoxynivalenol is the most common trichothecene affecting
agriculture production in the US, usually produced by Fusarium
graminearum, and causing major losses in maize, wheat, barley and
other small grains. The presence of rain during grain flowering
encourages fungus dispersal and infection. Areas of the Midwest and
the Red River Valley are particularly affected by the disease. The
presence of the fungus greatly reduces grain yields, and a very
small amount of fungal contamination renders barley unusable for
malting.
Methods
[0039] The activities of lichen extracts were evaluated using
experimental fungal organisms to ascertain whether the extracts
could inhibit the growth or functions of the fungal organisms. In
experiments described herein tested for their effects on
sporulation, hyphal growth and mycotoxin production in the
filamentous fungus Aspergillus parasiticus. For example,
Aspergillus parasiticus is one of the main producers of
aflatoxin.
[0040] Aspergillus parasiticus is useful for screening for
antifungal activity. A. parasiticus is a plant pathogen, and
produces the mycotoxin aflatoxin. It is extremely closely related
to A. flavus, a human and plant pathogen, and A. fumigatus, a human
pathogen. Vegetative growth allows fungi to colonize substrates,
such as grain, and human tissues in the case of mycotic disease.
Vegetative growth is associated with the production of toxins such
as aflatoxin. Sporulation is important in dissemination of fungi in
open air, agricultural fields, and in systemic spread through the
human body. Small spores are easily carried in the bloodstream.
Thus, compounds effective on A. parasiticus can be potentially used
in medical, agricultural and food safety applications. A primary
visual screen of growth, sporulation and secondary metabolism has
been performed on lichen extracts using a mutant of A. parasiticus
called B62 that accumulates norsolorinic acid, an orange-red
precursor of aflatoxin (FIG. 2). As illustrated herein, use of this
species for assay screens permits quick identification of lichen
extract activities.
[0041] Also as illustrated herein, Fusarium graminearum can be used
in assays for screening lichen extracts for inhibiting
deoxynivalenol production by the Fusarium species.
[0042] Lichen extracts can be prepared in a variety of ways. In
some embodiments, lichen samples are dried (e.g. at room
temperature in the dark). Samples can then be ground in a mill.
Samples are then extracted with a solvent (e.g., an alcohol, ethyl
acetate, or acetone). Solvents other than alcohols, ethyl acetate,
and acetones were not as effective. The solvent employed for
extraction can be methanol, ethanol ethyl acetate, acetone, or a
combination thereof.
[0043] In some embodiments, extracts are then filtered and dried.
The resulting residue can be dissolved in a solvent (e.g., alcohol,
ethyl acetate, acetone, water or a combination thereof) to yield a
final suspension. In some embodiments, suspensions are generated
with different concentrations of lichen material. In some
embodiments, the extract preparation methods described in Examples
1-5 below are utilized.
[0044] In some embodiments, active ingredients (e.g., with
anti-fungal activity or toxin inhibitory activity) are further
purified. Purification methods are well known in the art and
include, but are not limited to, extraction, fractionation, and
chromatography. The presence of active ingredient is followed at
each step of the process (e.g., using the activity assays described
herein) and fractions with active ingredients are carried to the
next step.
[0045] In some embodiments, purified active components are
identified. Methods for identifying both small molecule and large
molecule (e.g., protein) components are available and include, but
are not limited to, high pressure liquid chromatography (HPLC),
mass spectroscopy, nuclear magnetic resonance, and combinations
thereof.
[0046] The experiments described herein illustrate that lichen
extracts are useful as inhibitors of fungal growth and fungal
functions. For example, several lichen extracts and pure lichen
compounds exhibit inhibitory effects on aflatoxin production by
Aspergillus parasiticus, when grown in culture or on corn kernels,
as a model for field conditions. Specifically, pre-treatment of
corn kernels with crude extracts of Cladonia cristatela,
Pseudevernia firfuracea, Cetraria islandica, Cetrelia olivetorum,
or Evernia prunastri inhibited aflatoxin BI accumulation when
applied prior to inoculation with A. parasiticus. Extracts of C.
cristatela showed significant inhibition of AFB I by 99%. Lecanoric
acid, identified in the extract of Cetrelia olivetorum, and
methyl-orsellinate, which is present in Physcia stellaris and
Hypogymnia physodes, demonstrated inhibitory effects on growth and
aflatoxin production in A. parasiticus culture.
[0047] Extracts of 70 lichen species were generated. Three of the
seventy extracts inhibited Aspergillus parasiticus growth. Ten of
the seventy extracts inhibited Aspergillus parasiticus sporulation.
Fifty-nine of the lichen extracts inhibited secondary metabolite
production, where aflatoxin was used as a proxy for secondary
metabolite production. The most common activity observed for the
lichen extracts across the phylogeny was therefore in arresting
secondary metabolite production.
[0048] Extracts of two lichens were tested in cultures with
Fusarium graminearum, using assay methods like those described for
Aspergillus parasiticus. Treated cultures showed significant
reduction in deoxynivalenol and 1-acetyldeoxynivalenol without
significant effect on Fusarium graminearum growth.
Compositions
[0049] Compositions described herein can include at least one
lichen extract, extract component, or compound purified from a
lichen extract. The compositions can also include additional
components such as a carrier, solvent, surfactant, an additional
active ingredient, or a combination thereof.
[0050] These compositions are useful as antibiotics and anti-fungal
compositions.
[0051] The composition can contain varying amounts of the extract
components, or compounds purified from the extracts described
herein. For example, the extracts or compounds can be present in
liquid compositions at concentrations of about 0.1 .mu.g/mL to
about 1000 .mu.g/mL, or about 1 .mu.g/mL to about 800 .mu.g/mL, or
about 3 .mu.g/mL to about 600 .mu.g/mL, or about 5 .mu.g/mL to
about 500 .mu.g/mL, or about 5 .mu.g/mL to about 300 .mu.g/mL.
[0052] In dry compositions, the extracted components or compounds
can be present in at weight/weight concentrations of about 0.1
.mu.g/g to about 1000 .mu.g/g, or about 1 .mu.g/g to about 800
.mu.g/g, or about 3 .mu.g/g to about 600 .mu.g/g, or about 5
.mu.g/g to about 500 .mu.g/g, or about 5 .mu.g/g to about 300
.mu.g/g.
[0053] The compositions can therefore be dry compositions or liquid
compositions.
[0054] In some instances, the extracted components or compounds are
dissolved in a solvent to form a liquid composition with a known
concentration of at least one component or compound from an extract
described herein. The solvent can be an alcohol, ethyl acetate,
acetone, water, or a combination thereof. For example, the solvent
can be ethanol methanol, ethyl acetate, acetone, water, or a
combination thereof.
[0055] The compositions can contain a carrier such as an
emulsifier, a dispersing agent, thickening agent, a surfactant, a
clay, a polymer, a colorant, a wetting agent of ionic or non-ionic
type, a natural or regenerated mineral substance, a dispersant, a
wetting agent, a tackifier, a thickener, a binder, or a mixture of
such carriers. For example, the compositions can contain
polyacrylic acid salts, lignosulphonic acid salts, phenolsulphonic
or naphthalenesulphonic acid salts, polycondensates of ethylene
oxide with fatty alcohols or with fatty acids or with fatty amines,
substituted phenols (in particular alkylphenols or arylphenols),
salts of sulphosuccinic acid esters, taurine derivatives (in
particular alkyl taurates), phosphoric esters of polyoxyethylated
alcohols or phenols, fatty acid esters of polyols, and derivatives
of the present compounds containing sulfate, sulfonate and
phosphate functions. The presence of at least one surfactant can be
included when the active compound and/or the inert support are
water-insoluble and when the vector agent for the application is
water. For example, surfactant content can be about 5% to 40% by
weight of the composition.
[0056] Coloring agents such as inorganic pigments can be present in
the composition, for example iron oxide, titanium oxide, ferrocyan
blue, and organic pigments such as alizarin, azo and
metallophthalocyanine dyes, and trace elements such as iron,
manganese, boron, copper, cobalt, molybdenum and zinc salts can be
used. The compounds can be present in paints along with any
available coloring material(s) and other components typically
employed in paints.
[0057] Optionally, other additional components may also be
included, e.g. protective colloids, adhesives, thickeners,
thixotropic agents, penetration agents, stabilizers, sequestering
agents.
[0058] The compositions can also include other ingredients. For
example, bactericidal compounds can be employed. In addition, the
compounds described herein can be used together in a composition or
they can be used concomitantly with one or more of the other
agrichemicals such as various pesticides, acaricides, nematicides,
other types of fungicides, and plant growth regulators.
[0059] Various types of additional fungicides can optionally be
included in the compositions described herein. Examples include
copper fungicide such as basic copper chloride and basic copper
sulfate, sulfur fungicide such as thiuram, zineb, maneb, mancozeb,
ziram, propineb, and polycarbamate, polyhaloalkylthio fungicide
such as captan, folpet, dichlorfluanid, organochlorine fungicide
such as chlorothalonil, fthalide, organophosphorous fungicide such
as O,O-bis(1-methylethyl)S-phenylmethyl phosphorothioate (IBP),
edifenphos (EDDP), tolclophos-methyl, pyrazophos, fosetyl,
dicarboxylmide fungicide such as iprodionc, procymidone,
vinclozolin, fluoromide, carboxyamide fungicide such as
oxycarboxin, mepronil, flutolanil, tecloftalam, trichlamide,
pencycuron, acylalanine fungicide such as metalaxyl, oxadixyL
furalaxyl, methoxyacrylate fungicides such as kresoxim-methyl
(stroby), azoxystrobin, metominostrobin, trifloxystrobin,
pyraclostrobin, anilinopyrimidine fungicide such as andupurine,
mepanipyrim, pyrimethanil, cyprodiniL antibiotic agents such as
polyoxin, blasticidin S, kasugamycin, validamycine,
dihydrostreptomycin sulfate, propamocarb hydrochloride, quintozene,
hydroxyisoxazole, methasulfocarb, anilazine, isoprothiolane,
probenazole, chinomethionat, dithianon, dinocap, diclomezine,
ferimzone, fluazinam, pyroquilon, tricyclazole, oxolinic acid,
iminoctadine acetate, iminoctadine albesilate, cymoxanil,
pyrrolnitrin, diethofencarb, binapacryl, lecithin, sodium
bicarbonate, fenaminosulf, dodine, dimethomorph, phenazine oxide,
carpropamide, flusulfamide, fludioxonil, famoxadone, or
combinations thereof. Hence, other types of fungicides can be mixed
together with and used in various amounts with one or more of the
extracts or compounds described herein.
[0060] The extracts, extract components, and compounds described
herein can be used in a weight ratio relative to the other type of
fungicide such as from 1:0.001 to 1:1000 as a weight ratio. In some
instance, the amount of an extract, extract component, or compound
purified from a lichen extract relative to the other type of
fungicide can vary from 1:0.01 to 1:100 as a weight ratio within a
composition.
[0061] Pesticides can be included in the compositions, with any of
the compounds described herein. The pesticides can include
organophosphorous pesticides, carbamate pesticides such as
fenthion, fenitrothion, diazinon, chlorpyrifos, ESP, vamidothion,
phenthoate, dimethoate, formothion, malathon, trichlorfon,
thiometon, phosmet, dichlorvos, acephate, EPBP, methylparathion,
oxydemeton-methyl, ethion, salithion, cyanophos, isoxathion,
pyridaphenthion, phosalone, methidathion, sulprofos,
chlorfevinphos, tetrachlorvinphos, dimethylvinphos, propaphos,
isofenphos, ethylthiometon, profenofos, pyraclofos, monocrotophos,
azinphosmethyl, aldicarb, methomyl, thiodicarb, carbofuran,
carbosulfan, benfuracarb, furathiocarb, propoxur, BPMC, MTMC, MIPC,
carbaryl, pirimicarb, ethiofencarb, and fenoxycarb, pyrethroid
pesticides such as permethrin, cypermethrin, deltamethrin,
fenvalerate, fenpropathrin, pyrethrin, allethrin, tetramethrin,
resmethrin, dimethrin, propathrin, phenothrin, prothrin,
fluvalinate, cyfluthrin, cyhalothrin, flucythrinate, ethofenprox,
cycloprothrin, tralomethrin, silafluofen, brofenprox, and
acrinathrin, and benzoylurea and other types of pesticides such as
diflubenzuron, chlorfluazuron, hexaflumuron, triflumuron,
tetrabenzuron, flufenoxuron, flucycloxuron, buprofezin,
pyriproxyfen, methoprene, benzoepin, diafenthiuron, acetamiprid,
imidacloprid, nitenpyram, fipronil, cartap, thiocyclam, bensultap,
nicotin sulfate, rotenone, mataldehyde, machine oil, and microbial
pesticides e.g. BT and insect pathogenic virus.
[0062] Acricides can be included in the compositions described
herein. The acricides that can be employed include, for example,
chlorbenzilate, phenisobromolate, dicofol, amitraz, BPPS,
benzomate, hexythiazox, fenbutatin oxide, polynactin,
chinomethionat. CPCBS, tetradifon, avermectin, milbemectin,
clofentezin, cyhexatin, pyridaben, fenpyroximate, tebufenpyrad,
pylidimifen, fenothiocarb, and dienochlor.
[0063] As for the aforementioned nematicides, fenamiphos,
fosthiazate and the like can be specifically exemplified; as for
plant-growth regulators, gibberellins (e.g., gibberellin A3,
gibberellin A4, and gibberellin A7), auxin, 1-naphthaleneacetic
acid, and so on can be specifically exemplified.
[0064] More generally, the active compounds can be combined with
any solid or liquid additive, which complies with the usual
formulation techniques. In general, the composition according to
the invention may contain from 0.05 to 99% by weight of active
compounds, preferably from 10 to 70% by weight.
[0065] The extracts, extract component, extracted compounds or
compositions can be provided in a form that is ready-to-use or in a
form that can be prepared for use. The extracts, extract component,
extracted compounds, or compositions can be applied by a suitable
device, such by use of a spraying or dusting device. The extracts,
extract component, extracted compounds, or compositions can be
applied by use of brush or roller.
[0066] The extracts, extract component, extracted compounds, or
compositions can be provided in concentrated commercial
compositions that should be diluted before application to the crop.
For example, the extracts, extract component, extracted compounds,
or compositions can be provided in dry (e.g., lyophilized) form, or
in concentrated form, and then dissolved or diluted as desired. The
compositions can be in formulated into an aerosol dispenser, as a
capsule suspension, as a cold fogging concentrate, as a dustable
powder, as an emulsifiable concentrate, as an emulsion oil in
water, as an emulsion water in oil, as an encapsulated granule, as
a fine granule, as a flowable concentrate for seed or nut
treatment, as a gas (under pressure), as a gas generating product,
as granules, as a hot fogging concentrate, as macrogranules, as
microgranules, as an oil dispersible powder, as an oil miscible
flowable concentrate, as an oil miscible liquid, as a paste, as a
plant rodlet, as a powder for dry seed or nut treatment, as seeds
or nuts coated with the composition, as a soluble concentrate, as a
soluble powder, as a solution for seed (or other) treatment, as a
suspension concentrate (flowable concentrate), as an ultra-low
volume (ULV) liquid, as an ultra-low volume (ULV) suspension, as
water dispersible granules or tablets, as a water dispersible
powder for slurry treatment, as water soluble granules or tablets,
as a water soluble powder for seed or nut treatment, as a wettable
powder, or as a combination thereof (e.g., two types of
formulations packaged together).
[0067] The following Examples illustrate some of the experimental
work involved in the development of the invention.
Example 1: Materials and Methods
[0068] This Example describes materials and methods for extraction
of lichens. Lichens were collected fresh, and in some cases
herbarium specimens were surveyed, where the oldest herbarium
specimen dated back to 1949. Extracts were done in methanol,
ethanol, ethyl acetate, and acetone; other solvents did not appear
to extract activities as well. Activities were found to be quite
stable in herbarium specimens, even amongst the oldest
specimens.
[0069] A primary visual screen of growth, sporulation and secondary
metabolism was performed on lichen extracts using a mutant of
Aspergillus parasiticus strain B62 which accumulates norsolorinic
acid (NA), an orange-red precursor of aflatoxin. Norsolorinic acid
quantification was performed by use of a chromameter. Use of this
species for initial screens has facilitated fast identification of
activities.
[0070] Extracts of two lichens were tested in cultures with
Fusarium graminearum, using methods like the assay methods used for
Aspergillus parasiticus. Aspergillus parasiticus was grown on
glucose mineral salts (GMS) medium. Fusarium graminearum was grown
on High DON Medium from Linda Harris (McCormick et al., Appl.
Environ. Microbiol. 70:2044-2051 (2004)).
Example 2: Aspergillus parasiticus Strain B62 Treated with Lichen
Extracts
[0071] A set of high-throughput screens for discovery of antifungal
agents were developed using spices as a potential source of
inhibitors of mycotoxin biosynthesis. Black pepper extracts were
used in an initial screen.
[0072] Extracts of spices were added to Aspergillus parasiticus B62
cultures in GMS, YES, and PMS media (Buchanan & Lewis (1984))
as described in Annis et al. (2000). Changes in growth, sporulation
and mycotoxin production were observed. Extracts with promising
effects were screened using the Thin Layer Chromatography (TLC)
bioassay developed by the inventors and coworkers (Annis et al.,
2000; Trail et al. 2006). This technique resulted in the
identification of a novel activity in black pepper that turned off
genes for aflatoxin production but did not arrest fungal growth.
Based on HPLC, and MS analysis, putative structures were
synthesized and two active structures have been identified. One
structure inhibits aflatoxin biosynthesis but does not affect
fungal growth, the other inhibits fungal growth.
[0073] In preliminary studies, extracts from four lichen species
were tested for effects on growth, sporulation and mycotoxin
biosynthesis in A. parasiticus (FIG. 2). Extracts from one lichen
species inhibited fungal growth, and extracts from a second lichen
greatly reduced aflatoxin accumulation, but did not affect growth,
and inhibited sporulation. These preliminary trials indicate a high
frequency of bioactive compounds in a small group of randomly
selected lichens.
[0074] High through-put assays were performed in 24 well plates to
evaluate the antifungal effects of lichen extracts on the fungal
Aspergillus parasiticus strain B62. An orange-red pigment was a
precursor and indicator of aflatoxin production.
[0075] FIG. 2A-2E illustrate high through-put assays in 24 well
plates for antifungal effects of lichen extracts on the fungus
Aspergillus parasiticus strain B62. FIG. 2A shows a 3-day old
culture of Aspergillus parasiticus strain B62 that was not treated
(control). FIG. 2B shows a 3-day old culture of Aspergillus
parasiticus strain B62 treated with an aflatoxin biosynthesis
inhibitor. FIG. 2C shows a 3-day old culture of Aspergillus
parasiticus strain B62 treated with a growth inhibitor. FIG. 2D
shows a 7-day old culture of Aspergillus parasiticus strain B62
illustrating spore production (control). FIG. 2E shows a 7-day old
culture of Aspergillus parasiticus strain B62 treated with a
sporulation inhibitor. Plates with 24 wells were used for these
assays.
Example 3: Assay Results
[0076] Table 1 shows results of assays for inhibition of growth,
aflatoxin production, and sporulation of Aspergillus parasiticus
B62 by extracts of the indicated lichen species are shown. The
growth, aflatoxin production, and sporulation of Aspergillus
parasiticus B62 was compared to Control. The symbols used in Table
1 were: N--No effect; SI--Strong Inhibition (90-100%);
I--Inhibition (38-80%); A--Activation (.about.150%); SA--Strong
Activation (>150%).
TABLE-US-00001 TABLE 1 Effects of Lichen Extracts on growth,
aflatoxin production, and sporulation of Aspergillus parasiticus
B62 Lichen species Growth Aflatoxin Sporulation Bryoria fremontii N
SI N Cetraria islandica N SI N Latvia May 2016 (I Fract) Cetrelia
olivetorum CA Bay Area August 2016 Cetrelia sp CA Bay Area August
2016 Cladonia chlorophaceae N I N Pellston MI 3 Oct. 2015 Cladonia
crispata Pellstone MI 17 Sep. 2016 Cladonia cristatela (British
soldier) N SI N Frances 17 Nov. 2015 Red Caps/Rotten Cart Cladonia
didyma + N SI A Cladonia fimbriata Cladonia digitata N SI N Latvia,
Lielupe, 9 Aug. 2015 Cladonia mitis Pellstone MI 17 Sep. 2016
Cladonia pyxidata (Owen) N I N Cladonia rangiferina N I A Latvia
May 2016 Ropazi Maris Small quantity Cladonia stellaris N SI I
Yoshkar-Ola 2009 Cladonia sylvatica I SI I Yoshkar-Ola 2009
Dendrographa leucophaea N SI N f. minor Frances March CA 2017
Dermatocarpon miniatum N SI N S Dakota Black Hills on rock
Herbarium Ephebe lanata N SI N Flora Suecica 1961 Herbarium Evernia
mesomorpha N SI N? Pellston (#3) 3 Oct 2015 Evernia prunastri SI SI
SI Yoshkar-Ola 2009/GB 2015 Flavopanctelia flaventior N SI I by
Green House 19 Jun. 2015 Flavoparmelia caperata N SI I by Green
House 19 Jun. 2015 Flavopunctelia soredica N SI N Quercus alba
tree, F.T., 8 Aug 15 Fuscopannaria leucosticta N N N N Carolina
1967 Herbarium Graphis bulacana N SI N West Indies Trinidad 1963
Herbarium Herpothalon rubrocinctum 2016 Hypogymnia physodes I SI SI
Latvia, Lielupe, 9 Aug. 2015 Lecanora caesiorubella CA March 2017
Lecanora circumborensis CA March 2017 Lecidea psora N SI Wyoming
1956 Herbarium Lepraria lohiticans N "Amorphic" Dust Lichen
Pellston MI 3 Oct 2015 Leptogium saturninum N I S Dakota Alan 1961
Letharia vulpina, N SI N wolf lichen, Alan, 1994 Lobaria pulmonaria
Maine Frances 10-15 Jul. 2016 N Nephroma arctica N SI MSC0066318
Niebla cephalota N SI N Asilomar 2015 Parmelia sulcata + N SI N
Flavoparmelia caperata Parmeliella plumbea N SI N Canary Islands
Tenerife 1964 Herbarium Peltigera canina N I/N N Pellston MI 3 Oct.
15, (#10) Peltigera trumlata AF10981 N I N Peltula euploca N SI A
Colorado Rabit Mt 1960 Herbarium Pertusaria erebescens N N N East
Falklands Kidney Is1, 1968 Herbarium Phaeophyscia orbicularis, Alan
N I N Physcia aipolia Okemos 2014 N SI N Physcia millegrana N SI SA
Frances' Yard 22 Jun. 2015 Physcia stellaris N SI I by Vet Clinic
19 Nov. 2015 Physconia deters N I I MI Alan 8 Oct. 1976 Platismatia
glauca N SI I Latvia May 2016 Ropazi Maris/Ilze Pseudevernia
farinacea N(I/SI SI N Latvia May 2016 Ropazi Maris/Ilze Fracts) (P.
furfuracea by ITS/NCBI) Pseudevernia furfuracea I SI N Latvia,
Lielupe, 9 Aug 2015 Pseudocyphellaria berberina N N SI AF10717
Pseudocyphellaria crocata AF 10876 N SI N Pseudocyphellaria
freycinetii AF N SI I(?) 10716 Pseudocyphellaria gilva, AF 10877 N
SI N Pseudocyphellaria norvegica AF N I N 10878 Ramalina farinacea
Asilomar March N N N 2015 Ramatina fastigiata N SI N Latvia,
Lielupe, 9 Aug. 2015 Ramalina fraxinea Latvia May 2016 N I I
Carnicava Skaidrite Ramalina menziesii Asilomar 2015 N SI A(one
well) Rhizoplaca melanophthalma N N N S Dakota Black Hills 1960
Herbarium Roccella portentosa N SI N Chile Alan 14 Nov. 1965
Stereocaulon saxatile MI Cheboygan N SI N County 1949 Herbarium
Thamnolia vermicularis N SI N British Columbia 1970 Herbarium
Umbilicaria hyperborea + N I N Pseudephebe pubescens Usnea
arizonica Maine July 2016 Frances Usnea rubicunda N N N Asilomar
March 2015 Usnea strigosa N I N Usnea subfloridana N SI N Usnea
wasmathii SI Latvia May 2016 Ropazi Maris/Ilze Small quantity
Xanthoparmelia chlorochroa A SI Xanthoria hasseana N I SA yellow
Pellston (#3) 3 Oct. 2015 Xanthoria parietina N SI N?
Example 4: Cladonia cristatella Extracts Inhibit Secondary
Metabolite Production
[0077] This Example illustrates that extracts of Cladonia
cristatella inhibits secondary metabolite production by Aspergillus
parasiticus strain B62.
[0078] Ascospores from Cladonia cristatella were isolated and
cultured as illustrated in FIG. 3A. Colonies from these colonies
were extracted with ethanol and acetone. Aspergillus parasiticus
strain B62 was cultured with the extracts. As illustrated in FIG.
3B, the extracts inhibited secondary metabolite production by
Aspergillus parasiticus strain B62.
Example 5: Evernia prunastri Extracts Inhibit Hyphal Growth,
Secondary Metabolite Production, and Sporulation
[0079] This Example illustrates that extracts of Evernia prunastri
inhibit hyphal growth, secondary metabolite production, and
sporulation by Aspergillus parasiticus strain B62.
[0080] Extracts of Evernia prunastri were cultured with Aspergillus
parasiticus B62 in GMS (Buchanan & Lewis, 1984). As illustrated
in FIG. 4B the Evernia prunastri extracts exhibited strong
inhibitory activity against hyphal growth, secondary metabolite
production and sporulation by Aspergillus parasiticus B62.
Purification of the active compound and X-ray crystallography
determined that evemic acid was present in active fraction of
Evernia prunastri extracts, where the evemic acid structure is
shown below.
##STR00001##
Example 6: Lichen Extracts Inhibit Accumulation of Aflatoxins in
Corn Kernels
[0081] The following lichen extracts were tested for their capacity
to reduce aflatoxin accumulation in corn kernels.
[0082] #1 Evernia prunastri
[0083] #81 Cladonia cristatella
[0084] #99 Pseudevernia farinacea
[0085] #101 Cetraria islandica
[0086] #129 Dendrographa leucophaea f. minor
[0087] Extracts of lichens #1, 99 and 101 demonstrated protective
effects against aflatoxin contamination, showing 80-70% inhibition
of aflatoxin accumulation on DeDell field corn kernels (200
spores/kernel, 96 h post inoculation). Extracts obtained from
lichen #81 inhibited aflatoxin B1 accumulation by 99% on sweet corn
kernels. The extract from #129 was active on Monsanto field corn
kernels after 72 h post inoculation. All of these represent
statistically significant findings.
Example 7: Stability of Compounds in Lichen Material During Storage
and Use of Herbarium Specimens for Extraction
[0088] Chemicals preserved in dry lichens appear to be largely
stable under standard herbarium storage conditions. Thin layer
chromatography (TLC) analyses of acetone extracts from the
identical lichen specimens obtained eight years apart demonstrated
similar metabolite profiles (FIG. 5A-5B).
Example 8: Lichen Extracts Reduce 1-Acetyldeoxynivalenol by
Fusarium graminearum
[0089] Extracts of two lichens. Evernia pruastris and Hypogymnia
physodes, were tested in cultures with Fusarium graminearum, using
assay methods like those used for the assays of Aspergillus
parasiticus.
[0090] As illustrated in FIG. 6, the Fusarium graminearum cultures
treated with these lichen extracts (Evernia pruastris [number 1],
and Hypogymnia physodes [number 57]) showed significant reduction
in 1-acetyldeoxynivalenol production without significant effects on
growth.
Example 9: Extracts of Various Lichen Reduce Deoxynivalenol
Production
[0091] The fungus Fusarium graminearum is a plant pathogen which
causes fusarium head blight, a devastating disease on wheat and
barley. This Example illustrates that extracts from various lichens
can reduce deoxynivalenol (DON) production by Fusarium
graminearum.
[0092] In a first experiment. Fusarium graminearum cultures were
grown in 24-well plates for 6 days in the dark at room temperature.
Each well contained 1 ml High DON Medium (McCormick et al., 2004)
plus 20 .mu.l of lichen extract (either Evernia pruastris [number
1] or Hypogymnia physodes [number 57]), while control wells
contained 20 .mu.l ethanol without a lichen extract. Cultures
exhibiting decreased production of red/pink pigment but no effects
on growth were analyzed for DON accumulation. FIG. 7A shows the
effects of these lichen ethanol extracts on DON accumulation by
Fusarium graminearum grown in culture for 6 days.
[0093] In a second experiment. Fusarium graminearum cultures were
grown in 24-well plates for 6 days in the dark at room temperature.
Each well contained 1 ml High DON Medium plus 20 .mu.l of a lichen
extract from Cladonia sylvlaica [15], Usnea strigosa [33], Cladonia
digitate [58], Ramalina fastigiate [62], Ramalina fraxinea [98],
Platismatia glauca [100], Evernia prunastri [112], or Cladonia
crispata [118]. Control wells contained 20 .mu.l acetone without
any lichen extract. Cultures exhibiting decreased production of
red/pink pigment but no effects on growth were analyzed for DON
accumulation.
[0094] As shown in FIG. 7B acetone extracts of lichen extracts from
Cladonia sylvlaica [15], Usnea strigosa [33], Cladonia digitate
[58], Ramalina fastigiate [62], Ramalina framinea [98], Platismatia
glauca [100], Evernia prunastri [112], and Cladonia crispala [118]
all reduced DON production by Fusarium graminearum.
Example 10: Extracts of Various Lichen Remain Stable for
Decades
[0095] This Example described results of assaying various lichens
that had been stored for extended periods of time to determine if
they could still inhibit growth, norsolorinic acid (NA) production,
and sporulation by Aspergillus parasiticus.
[0096] Lichens were obtained after storage under herbarium storage
conditions, extracts were made, and the extracts were added to
Aspergillus parasiticus cultures.
[0097] This assessment of lichen extract activity was performed in
2014-2015.
[0098] Table 2 shows which lichens were evaluated, and whether the
Aspergillus parasiticus grew, accumulated norsolorinic acid, and
sporulated (N indicates that no growth, no NA, or no sporulation
was observed).
TABLE-US-00002 TABLE 2 Activity of lichen compounds to inhibit
Norsolorinic Acid accumulation is stable for decades under
Herbarium storage conditions. Inhibition of Lichen/ Norsolorinic ID
Year collected/ acid number Storage facility Growth accumulation
Sporulation 85 Stereolaulon saxatile, N 90-100% N Michigan
Cheboygan County 1949, MSU Herbarium 87 Petula euploca, N 90-100% N
Colorado Rabbit Mt, 1960, MSU Herbarium 88 Graphis bulacana, N
90-100% N West Indies, Trinidad, 1963, MSU Herbarium 90 Ephebe
lanata, Flora N 90-100% N Suecica, 1961, MSU Herbarium 93 Lecidea
psora, N 90-100% N Wyoming, 1956, MSU Herbarium
Activity of assay was performed in 2014-2015. N, no effect.
[0099] As illustrated, each of the lichens shown in Table 2
inhibited 90-100% of norsolorinic acid accumulation even though the
lichens had been stored since 1949-1963.
REFERENCES
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[0119] All patents and publications referenced or mentioned herein
are indicative of the levels of skill of those skilled in the art
to which the invention pertains, and each such referenced patent or
publication is hereby specifically incorporated by reference to the
same extent as if it had been incorporated by reference in its
entirety individually or set forth herein in its entirety.
Applicants reserve the right to physically incorporate into this
specification any and all materials and information from any such
cited patents or publications. The following statements describe
some of the elements or features of the invention. Because this
application is a provisional application, these statements may
become changed upon preparation and filing of a nonprovisional
application. Such changes are not intended to affect the scope of
equivalents according to the claims issuing from the nonprovisional
application, if such changes occur. According to 35 U.S.C. .sctn.
111(b), claims are not required for a provisional application.
Consequently, the statements of the invention cannot be interpreted
to be claims pursuant to 35 U.S.C. .sctn. 112.
Statements:
[0120] 1. A method comprising extracting a lichen sample with
alcohol, ethyl acetate, acetone or a combination thereof to provide
a lichen extract, and measuring whether the lichen extract inhibits
the growth, sporulation, or mycotoxin production of a fungus.
[0121] 2. The method of statement 1, wherein the fungus produces
aflatoxin. [0122] 3. The method of statement 1 or 2, wherein the
fungus is an Aspergillus or a Fusarium. [0123] 4. The method of
statement 1, 2, or 3, wherein the fungus is an Aspergillus
parasiticus. [0124] 5. The method of statement 1-3 or 4, wherein
the fungus is Aspergillus parasiticus strain B62. [0125] 6. The
method of statement 1-4 or 5, wherein the fungus is Fusarium
graminearum. [0126] 7. The method of statement 1-5 or 6, wherein
the alcohol is methanol or ethanol. [0127] 8. The method of
statement 1-5 or 6, wherein the lichen sample is extracted with
acetone. [0128] 9. The method of statement 1-7 or 8, further
comprising purification of a compound that inhibits the growth,
sporulation, or mycotoxin production of a fungus from the lichen
extract to provide a purified compound. [0129] 10. The method of
statement 1-8 or 9, further comprising determining a structure of a
compound that inhibits the growth, sporulation, or mycotoxin
production of a fungus from the lichen extract to provide a
compound with a structure. [0130] 11. The method of statement 1-9
or 10, further comprising determining an amount of the purified
compound or the compound with the structure that inhibits the
growth, sporulation, or mycotoxin production of a fungus. [0131]
12. The method of statement 1-10 or 11, further comprising applying
the lichen extract, the purified compound, or the compound with the
structure to a surface, an agricultural crop, a storage bin, a
storage facility, an animal feed, a plant seed, a nut, a plant
part, a plant product, or a combination thereof. [0132] 13. The
method of statement 1-11 or 12, wherein the surface is a bathroom,
a kitchen, a closet, a basement, an attic, an entryway, a cabinet,
a boat, a barn, an animal shelter, a warehouse, a grain storage
compartment, or a food storage facility surface. [0133] 14. The
method of statement 1-12 or 13, wherein the plants comprise
grain-producing plants, nut-producing plants, vegetable-producing
plants, fruit-producing plants, starch-producing plants,
fiber-producing plants, fodder-producing plants, grains, nuts,
vegetables, fruits, starch, fibers, flour, fodder, leaves, stock,
seeds, oil, or a combination thereof. [0134] 15. The method of
statement 1-13 or 14, wherein the plant products are almonds,
barley, betel nuts, brazil nuts, cashews, chestnuts, coconut,
coffee, corn, flour, hazelnuts, macadamia nuts, oats, pecans,
peanuts, pine nuts, pistachios, rice, rye, sesame seeds, soybean,
spices, walnuts, wheat, or combinations thereof. [0135] 16. The
method of statement 1-14 or 15, wherein the mycotoxin content in a
surface, an agricultural crop, a storage bin, a storage facility,
an animal feed, a plant seed, a nut, a plant part, a plant product,
or a combination thereof is reduced by spraying a composition onto
the plants or plant products. [0136] 17. The method of statement
1-14 or 15, wherein the mycotoxin content is reduced by at least
10%, at least 20%, at least 30%, at least 40%, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%,
or at least 99%. [0137] 18. The method of statement 1-16 or 17,
wherein the lichen is any of the lichens described in this
application. [0138] 19. The method of statement 1-17 or 18, wherein
the lichen is Cladonia crispata. Cladonia cristatella, Cladonia
digitate, Cetraria islandica, Cetrelia olivetorum, Cladonia
svlvatica, Evernia pnsnastri, Hypogyrnia physodes, Platismatia
glauca, Pseudevernia fifuracea, Ranalina fastigiate, Ramalina
fraxinea, Usnea strigosa, or a combination thereof. [0139] 20. An
extract obtained by the method of statement 1-18 or 19. [0140] 21.
An extract component or compound obtained by the method of
statement 1-19 or 20. [0141] 22. An extract comprising lichen
compounds solubilized in alcohol, ethyl acetate, acetone or a
combination thereof. [0142] 23. A composition comprising a carrier
and an extract, compound, or a component of an extract obtained by
the method of statement 1-18 or 19. [0143] 24. The composition of
statement 23, where the carrier is a solvent or surfactant. [0144]
25. The composition of statement 23 or 24, where the carrier is a
solvent selected from methanol, ethanol ethyl acetate, or acetone.
[0145] 26. The composition of statement 23-24 or 25, where the
carrier is an emulsifier, a dispersing agent, a thickening agent, a
surfactant, a clay, a polymer, a colorant, a wetting agent, a
mineral substance, a dispersant, a tackifier, a thickener, a
binder, or a mixture of such carriers. [0146] 27. The composition
of statement 23-25 or 26, wherein the compound(s) are at a
concentration of 0.1 .mu.g/mL to about 1000 .mu.g/mL, or about 1
.mu.g/mL to about 800 .mu.g/mL, or about 3 .mu.g/mL to about 600
.mu.g/mL, or about 5 .mu.g/mL to about 500 .mu.g/mL, or about 5
.mu.g/mL to about 300 .mu.g/mL. [0147] 28. The composition of
statement 23-26 or 27, wherein the composition comprises
weight/weight concentrations of one or more compounds at about 0.1
.mu.g/g to about 1000 .mu.g/g, or about 1 .mu.g/g to about 800
.mu.g/g, or about 3 .mu.g/g to about 600 .mu.g/g, or about 5
.mu.g/g to about 500 .mu.g/g, or about 5 .mu.g/g to about 300
.mu.g/g. [0148] 29. The composition of statement 23-27 or 28,
wherein the compound(s) are at a concentration of about 0.1 ppm to
500 ppm, or about 1 ppm to 400 ppm, or about 2 ppm to 300 ppm, or
about 5 ppm to 250 ppm, or about 10 ppm to 150 ppm, or about 12 ppm
to 100 ppm, or about 15 to 50 ppm, or about 20 ppm to 35 ppm, or
about 25 ppm.
* * * * *