U.S. patent application number 14/027748 was filed with the patent office on 2014-03-27 for system for protection of plants from pathogens using alkamides.
This patent application is currently assigned to Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional (CINVESTAV). The applicant listed for this patent is Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional (CINVESTAV). Invention is credited to Luis Rafael Herrera-Estrella, Jose Lopez-Bucio, Jorge Molina-Torres.
Application Number | 20140088196 14/027748 |
Document ID | / |
Family ID | 41664029 |
Filed Date | 2014-03-27 |
United States Patent
Application |
20140088196 |
Kind Code |
A1 |
Lopez-Bucio; Jose ; et
al. |
March 27, 2014 |
SYSTEM FOR PROTECTION OF PLANTS FROM PATHOGENS USING ALKAMIDES
Abstract
System, including methods compositions, and kits, for
controlling the growth of fungi and other pathogens in plants using
at least one alkamide, which may function by increasing or
eliciting natural defense mechanisms of the plants against such
pathogens.
Inventors: |
Lopez-Bucio; Jose; (Morelia,
MX) ; Molina-Torres; Jorge; (Irapuato, MX) ;
Herrera-Estrella; Luis Rafael; (Irapuato, MX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Centro de Investigacion y de Estudios Avanzados del Instituto
Politecnico Nacional (CINVESTAV) |
Mexico City |
|
MX |
|
|
Assignee: |
Centro de Investigacion y de
Estudios Avanzados del Instituto Politecnico Nacional
(CINVESTAV)
Mexico City
MX
|
Family ID: |
41664029 |
Appl. No.: |
14/027748 |
Filed: |
September 16, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13057993 |
Feb 7, 2011 |
8536091 |
|
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PCT/IB2009/006677 |
Aug 5, 2009 |
|
|
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14027748 |
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61188058 |
Aug 5, 2008 |
|
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Current U.S.
Class: |
514/627 ;
514/625 |
Current CPC
Class: |
A01N 37/18 20130101;
A01N 37/20 20130101 |
Class at
Publication: |
514/627 ;
514/625 |
International
Class: |
A01N 37/20 20060101
A01N037/20 |
Claims
1. A method of plant protection, comprising: selecting a plant
susceptible to infection by, and/or infected with, a pathogen; and
applying an effective amount of at least one alkamide to the plant,
the effective amount being sufficient to inhibit growth of the
pathogen by inducing a defense response in the plant.
2. The method of claim 1, wherein the at least one alkamide is
applied in a composition, and wherein the concentration of the at
least one alkamide in the composition is less than about 10.sup.-6
M.
3. The method of claim 1, wherein the concentration of the at least
one alkamide in the composition is less than about 10.sup.-9 M.
4. The method of claim 1, wherein the alkamide has the formula
##STR00003## wherein R.sup.1 is hydrogen or has 1 to 8 carbons,
R.sup.2 is hydrogen or has 1 to 8 carbons, and R.sup.3 has 9 to 11
carbons.
5. The method of claim 4, wherein R.sup.1 has 4 carbons and R.sup.2
is hydrogen.
6. The method of claim 5, wherein the at least one alkamide is
N-isobutyl decenamide, N-isobutyl decanamide, or both.
7. A method of plant protection, comprising: selecting a plant
susceptible to infection by a fungus; and applying an effective
amount of N-isobutyl decanamide to the plant to inhibit growth of
the fungus.
8. The method of claim 7, wherein the fungus is member of the genus
Alternaria or Botrytis.
9. The method of claim 7, wherein the N-isobutyl decanamide, or an
oxidized precursor of the N-isobutyl decanamide, is extracted from
a natural source.
10. The method of claim 9, wherein the N-isobutyl decanamide or the
oxidized precursor is extracted from the natural source by
traditional solvent extraction or supercritical fluid extraction
(SFE).
11. The method of claim 9, wherein the N-isobutyl decanamide is
produced synthetically from the precursor.
12. The method of claim 11, wherein the precursor is affinin.
13. The method of claim 7, wherein the step of applying includes a
step of applying the effective amount as an aerosol, as a liquid
fertilizer composition, by dipping at least a portion of the plant
into a composition containing the N-isobutyl decanamide, or any
combination thereof.
14. The method of claim 7, wherein the plant is a seed and/or
seedling when application of N-isobutyl decanamide starts.
15. The method of claim 7, further comprising a step of applying at
least one other pathogen-controlling agent or defense elicitor to
protect the plant against attack by a pathogenic microorganism,
pest, and/or parasite.
16. The method of claim 7, further comprising a step of obtaining a
kit including a composition comprising the N-isobutyl decanamide
and configured for application to plants.
17. The method of claim 7, wherein the step of selecting selects a
plant already infected with the fungus.
18. The method of claim 7, wherein the step of applying includes a
step of contacting (a) a composition including N-isobutyl
decanamide and (b) one or more leaves, shoots, or roots of the
plant, or any combination thereof.
19. The method of claim 7, wherein the step of applying includes a
step of applying the N-isobutyl decanamide in a liquid or solid
formulation.
20. The method of claim 7, wherein the step of applying includes a
step of applying an amount of N-isobutyl decanamide sufficient to
kill the fungus.
Description
CROSS-REFERENCE TO PRIORITY APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/057,993, filed Feb. 7, 2011, now U.S. Pat.
No. 8,536,091, which in turn claims priority under 35 U.S.C.
.sctn.371 to PCT Patent Application Serial No. PCT/IB2009/006677,
filed Aug. 5, 2009, published Feb. 11, 2010, as WO 2010/015932,
which in turn claims priority to U.S. Provisional Patent
Application Ser. No. 61/188,058, filed Aug. 5, 2008. Each of these
priority documents is incorporated herein by reference in its
entirety for all purposes.
BACKGROUND
[0002] Agricultural production is under constant stress from global
population growth. Climate change, loss of arable land, and
pathogenic parasites (insects and microorganisms) continually
threaten agricultural production. New technologies, such as
creation of transgenic plants and genetic inbreeding, have helped
increase agricultural output, but are by no means a general
solution. The high cost of fuel, fertilizers, and pesticides have
also considerably increased the cost of food and endanger a
constant food supply for the developing world.
[0003] Plants continuously respond to abiotic and biotic stress by
adjusting their metabolism and activating diverse intracellular
signaling responses. In particular, modifications of plant
architecture in response to physical or chemical stimuli or the
activation of pathogen-specific defense mechanisms upon microbial
infection permits plant survival and reproduction. Classic
signaling molecules, such as auxin, cytokinin, gibberellins,
abscisic acid (ABA), and brassinosteroids, have been extensively
studied in the context of their role in morphogenetic processes,
whereas jasmonic acid (JA), ethylene, and salicylic acid (SA) have
been found to participate in defense-responses against
pathogens.
[0004] A novel group of lipid-derived plant metabolites, known as
alkamides, has been shown recently to alter plant metabolism and
development. Alkamides comprise over 200 related compounds that
have been found in as many as 10 plant families: Aristolochiaceae,
Asteraceae, Brassicaceae, Convolvulaceae, Euphorbiaceae,
Menispermaceae, Piperaceae, Poaceae, Rutaceae, and Solanaceae.
Plant-produced alkamides have been found to alter root and shoot
system architecture in Arabidopsis by affecting cell division and
differentiation processes. Morphogenetic responses affected by
alkamides included primary root growth, lateral and adventitious
root formation, root hair formation, and leaf development.
[0005] Alkamides have also been reported to have in vitro
antimicrobial activity, in the absence of plants. For example,
affinin has been demonstrated to inhibit the growth of some
microbial plant pathogens, including bacteria and fungi. However, a
reduced form of affinin, N-isobutyl decanamide, has been reported
to lack a fungistatic activity that is present in affinin. In any
event, relatively high levels of alkamides have been utilized to
demonstrate in vitro antimicrobial activity, potentially making
alkamides too expensive for large-scale use as antimicrobials.
[0006] Plants posses various inducible defense mechanisms to
protect themselves against pathogen attack. For example, systemic
acquired resistance (SAR) is activated in plants after infection by
necrotizing pathogens. Similarly, colonization of plant roots by
certain non-pathogenic rhizobacteria can elicit induced systemic
resistance (ISR) in the host plant. ISR is a plant-mediated
mechanism initiated at the root that extends up to the shoot.
Similar to SAR, ISR is effective against different types of plant
pathogens.
[0007] With the high cost and scarcity of food, increasing
agricultural production and efficiency is key worldwide. Control of
pathogens, especially bacteria and fungi, is important to enable
increased production of food and better use of fertile land, but
there is a growing awareness of the potential toxicity of standard
pesticides. Thus, organically grown produce and grains are becoming
important and growing markets. The use of natural, low cost
pesticides, such as pesticides that induce natural defense
mechanisms of plants, may significantly improve the outlook of the
food supply worldwide and may, at the same time, provide
eco-friendly pest control systems.
SUMMARY
[0008] The present disclosure provides a system, including methods,
compositions, and kits, for controlling the growth of fungi and
other pathogens in plants using at least one alkamide, which may
function by increasing or eliciting natural defense mechanisms of
the plants against such pathogens.
DETAILED DESCRIPTION
[0009] The present disclosure provides a system, including methods,
compositions, and kits, for controlling the growth of fungi and
other pathogens in plants using at least one alkamide, which may
function by increasing or eliciting natural defense mechanisms of
the plants against such pathogens.
[0010] Alkamides are a family of natural occurring compounds, which
upon isolation, or chemical synthesis, and subsequent application,
may strongly stimulate natural resistance in plants against
pathogenic fungi and bacteria (and other pathogens and parasites).
Thus, alkamides may become a new type of powerful, eco-friendly
pesticide for a wide variety of plants. This family of compounds
could act as powerful "defense elicitors" in plants and could have
great potential application in combating pathogenic pests.
Alkamides, such as N-isobutyl decanamide, can be key in triggering
adjustment of anatomical characteristics by the plant under attack,
enabling it to mount a better defense response, and additionally,
limiting its exposure to unfavorable environmental conditions.
[0011] The present disclosure presents data involving treatment of
Arabidopsis (A. thaliana) with N-isobutyl decanamide. The data show
that N-isobutyl decanamide induces many defense-related genes, in
addition to inducing genes potentially involved in growth and
development. The increased expression of defense response genes
occurs in parallel with accumulation of free and
glucoside-conjugated salicylic acid in seedlings. N-isobutyl
decanamide was found, when tested on Arabidopsis seedlings, to be
the most active alkamide identified to date. The data also show
that application of this compound to a plant confers resistance
against fungal necrotizing pathogens. When tested against the
necrotizing fungi Botrytis cinerea and Alternaria brassicicola,
N-isobutyl decanamide significantly reduced necrosis caused by the
pathogens and inhibited fungal proliferation on leaves when
compared to the controls. The data further show that N-isobutyl
decanamide can modulate salicylic acid-associated defense
responses, which lead to increased resistance against fungal
pathogens. Thus, alkamides like N-isobutyl decanamide represent a
new, biologically and ecologically friendly, system of control of
pathogenic fungi and other pathogens in plants using powerful,
eco-friendly pesticides.
[0012] The present disclosure provides a method of plant protection
using at least one member of a family of compounds, alkamides, as
natural pest controlling agents. These compounds may trigger or
stimulate high resistance in plants against pathogens. Alkamides
belong to a class of bioactive, amino lipids of wide distribution
in plants. The method may involve selecting a suitable plant and
applying an effective amount of an alkamide to the plant, as
described below.
[0013] At least one plant may be selected. The plant may be any
type of plant, at any stage of plant development (e.g., a seed,
seedling, mature plant, etc.). The plant also may be a isolated
portion of a plant, for example, in connection with plant
propagation (e.g., a bud, bulb, callus, corm, cutting, plantlet,
rhizome, rootstock, scion, stolon, tissue culture, tuber, etc.).
The plant may be susceptible to infection by a pathogen or may
already be infected by the pathogen. Exemplary pathogens that the
plant may be susceptible to and/or infected by include viruses,
bacteria, protozoans, fungi, and the like.
[0014] A composition comprising an alkamide may be applied to the
selected plant(s). The alkamide may have the following general
formula:
##STR00001##
R.sup.1 and R.sup.2 each, independently, may be hydrogen or an
organic moiety having 1 to 8 carbons, 1 to 6 carbons, or 4 carbons,
among others. In some embodiments, R.sup.1 may be isobutyl and
R.sup.2 may be hydrogen. R.sup.3 may be an organic moiety having 8
to 13 carbons, or 9 to 11 carbons (e.g., 9, 10, or 11 carbons),
optionally unsaturated at one, two, three, or more positions. The
carbonyl shown above is at position number 1, with the adjacent,
first carbon of R.sup.3 being at position number 2. If unsaturated,
R.sup.3 may be unsaturated at any one position or combination of
positions 2-10, such as one or more of positions 2, 4, 6, or 8,
among others, with each unsaturation being of the E or Z form. In
some embodiments, the alkamide may be affinin, which is
N-isobutyl-deca-2E,6Z,8E-trieneamide. In some embodiments, R.sup.3
has 9 carbons, and is unsaturated at position 2 only, such as in
the E form. For example, the alkamide may be
N-isobutyl-2E-decenamide. In some embodiments, the alkamide may be
N-isobutyl decanamide:
##STR00002##
[0015] The amount of alkamide applied to the selected plant(s) may
be an effective amount, that is, an amount sufficient to inhibit
pathogen growth, attack, and/or infection by inducing a defense
response to pathogens in the plant. A "defense response," as used
herein, is a response involving changes in gene expression in the
plant and resulting in an increased expression of one or more
defense-related plant genes (e.g., PRI) and/or increased levels of
one or more defense-related plant compounds, such as
free/conjugated salicylic acid and/or free/conjugated jasmonic
acid. An exemplary effective amount may be provided by an alkamide
disposed in a composition at a concentration of less than about
10.sup.-6 M, 10.sup.-7 M, 10.sup.-8 M, or 10.sup.-9 M, such as
about 10.sup.-6 to 10.sup.-12 M, or 10.sup.-8 to 10.sup.-12 M,
among others. The composition may be aqueous and/or may include one
or more salts, buffers, surfactants (ionic or nonionic), plant
nutrients/fertilizers, or plant hormones (one of or any combination
of an auxin, a cytokinin, a gibberellin, abscisic acid (ABA), a
brassinosteroid, jasmonic acid (JA), ethylene, and salicylic acid
(SA)), or any combination thereof.
[0016] The alkamide may be applied to the plant selected by any
suitable mechanism(s). The alkamide may be applied as a liquid or
solid formulation, among others. For example, the alkamide may be
sprayed onto the plant (such as in an aerosol or surface spray,
among others), applied to a growth medium (e.g., soil) in which
plant is disposed, spread on a portion of the plant, or the like.
Alternatively, or in addition, a portion or all of the plant may be
dipped in an alkamide composition. In an event, application of the
alkamide may include contacting (a) a composition including an
alkamide and (b) one or more leaves, shoots, or roots of the plant.
The application may include a seed treatment; foliar spray; soil
drench; pseudostem, root, stem, or trunk injection; fruit or root
dip; row treatment; etc.
[0017] The alkamide may be obtained from any suitable source(s) by
any suitable procedure or set of procedures. For example, the
alkamide, or an oxidized precursor of the alkamide, may be obtained
from a natural source, such as one or more plants. In some
embodiments, the alkamide or precursor may be isolated by solvent
extraction, supercritical fluid extraction, or a combination
thereof. Alternatively, or in addition, the alkamide or precursor
may be provided through organic synthesis, which optionally may be
catalyzed by one or more enzymes. In some cases, the alkamide may
be produced via chemical reduction, outside plants, of an oxidized
precursor, which, in turn, may have been isolated from plants.
Chemical reduction may, for example, be performed with hydrogen
gas, a catalyst (e.g., platinum), an enzyme, or a combination
thereof, among others.
EXAMPLE 1
N-Isobutyl Decanamide Affects Growth and Development of Arabidopsis
Seedlings without Compromising Cell Viability
[0018] One of the effects of alkamides on plants is the
dose-dependent inhibition of primary root growth. Experiments were
performed to validate the biological activity of
[0019] N-isobutyl decanamide on Arabidopsis seedlings prior to
characterization of global gene expression responses, and to
explore whether this inhibition of primary root growth could be due
to a developmental rather than to possible cytotoxic effects.
Arabidopsis seedlings were germinated for 6 d on agar plates
containing 0.2.times. Murashige and Skoog (MS) medium, and then
transferred to plates containing MS 0.2.times. agar medium supplied
with 60 pM N-isobutyl decanamide or solvent alone (as control).
Seven days after transfer (d.a.t.), plants exposed to the solvent
continued their normal growth, producing normal leaves and a
typical root system with proliferating lateral roots. In contrast,
N-isobutyl decanamide-treated seedlings showed inhibition of shoot
and root growth, greening of leaves, formation of increased numbers
of adventitious and lateral roots, and formation of callus-like
structures on leaf blades.
[0020] It was also determined that primary root growth inhibition
was not due to a toxic effect but rather to an effect of N-isobutyl
decanamide on cell division or elongation.
[0021] These results suggest that the effects of N-isobutyl
decanamide on root development involve changes in cellular
processes without affecting cell viability or integrity.
EXAMPLE 2
Transcription Profiling of N-Isobutyl Decanamide Effects
[0022] Transcription profiling of the effect of alkamides on
Arabidopsis seedlings was performed. N-isobutyl decanamide was
found to be the most active alkamide identified to date. A global
analysis of gene expression changes in response to N-isobutyl
decanamide treatment in Arabidopsis thaliana revealed 1,017
differentially regulated genes (579 were up-regulated and 437 genes
were down-regulated). When up-regulated genes were categorized by
function, it was observed that several gene families involved in
plant growth and development, and many others in plant defense
responses, were also up-regulated (including chitinases and plant
defensins and others).
EXAMPLE 3
N-Isobutyl Decanamide Induces Accumulation of Compounds Involved in
Defense Mechanisms
[0023] The levels of two important compounds involved in mediating
defense mechanisms in plants, salicylic acid (SA) and jasmonic
acid, were quantified after treatment with N-isobutyl decanamide. A
three- to four-fold increase in the relative levels of free SA, as
well as conjugated SA, was observed in N-isobutyl
decanamide-treated plants when compared to controls. This
experiment shows that N-isobutyl decanamide may stimulate defense
responses through these two compounds.
EXAMPLE 4
N-Isobutyl Decanamide Induces the Expression of PR1
[0024] Salicylic acid is known to induce plant resistance to a
number of pathogenic microbes in a process known as systemic
acquired resistance (SAR). The PR1 (pathogenesis-related 1) gene
has been implicated at least in part in the induction of disease
resistance in plants. Our gene expression analysis revealed a 2.5-,
4.3-, and 7.2-fold induction in PR1 (At2g14610) mRNA expression at
1 d, 3 d, and 7 d, respectively, after transfer of seedlings to
N-isobutyl decanamide-containing medium, suggesting an early
response of the PR1 gene (a plant defense marker) to this
alkamide.
[0025] Tests were performed to determine whether PR1 expression is
activated by N-isobutyl decanamide at the transcriptional level.
Transgenic A. thaliana seedlings containing a chimeric gene in
which the PR1 promoter is fused to the GUS reporter gene (PR1:GUS)
were treated with 60 pM N-isobutyl decanamide. GUS histochemical
determinations then were performed in 10 d-old seedlings. Control
seedlings did not show PR1:GUS expression in roots or shoots.
Interestingly, PR1:GUS expression was observed in sections of
shoots and roots in alkamide-treated seedlings. When detached
leaves from adult Arabidopsis plants were exposed to N-isobutyl
decanamide for 3 d, a uniform and high level of PR1:GUS was
detected in control leaves.
EXAMPLE 5
N-Isobutyl Decanamide Confers Resistance to Fungal Necrotrophic
Pathogens Botrytis cinerea and Alternaria brassicicola
[0026] Our results from microarray experiments showing SA
accumulation and PR1 induction suggest that N-isobutyl decanamide
may be detected by plants as a potential defense inducing factor
similar to those produced by avirulent pathogens leading to plant
resistance. To study whether N-isobutyl decanamide could
effectively activate defense mechanisms, we tested the response, of
leaves from 20 d-old Arabidopsis plants, to necrotrophic pathogens
Alternaria brassicicola and Botrytis cinerea. These pathogens
provoke spreading necrotic lesions on leaves. In these experiments,
fully developed leaves from 20 day-old plants were transferred to
medium supplied with N-isobutyl decanamide or with the solvent as
control. A. brassicicola and B. cinerea spores were inoculated on
the leaf surface and disease symptoms evaluated 3 d, 4 d, and 5 d
after inoculation (d.a.i.). In leaves transferred to control medium
and inoculated for 3 d, the two pathogens were found to induce
necrotic lesions in over 90% of inoculated leaves. On the other
hand, in leaves treated with N-isobutyl decanamide for 3 d, about
30% of leaves presented necrotic lesions caused by A. brassicicola
and only about 10% by B. cinerea attack.
[0027] It was also observed that lesions on untreated leaves were
about 6 mm in diameter, while lesions on leaves treated with
N-isobutyl decanamide were of a much smaller diameter, about
0.8-1.5 mm. Visual inspection showed that control leaves inoculated
with the pathogens had generalized necrotic lesions spanning half
or more the surface of the leaf at 4 d.a.i., while the
alkamide-treated leaves presented milder symptoms.
[0028] Next, growth of the pathogens was monitored by direct
microscopic observation of stained hyphae in infected leaves. It
was found that disease symptoms in control leaves at day 3 after
inoculation with A. brassicicola were accompanied by prolific
growth of fungal hyphae, which correlated with increased damage of
leaf tissue. However, N-isobutyl decanamide treatment inhibited
fungal development, resulting in decreased degradation of leaf
tissue. A similar protective effect of the alkamide could be
observed in leaves inoculated with B. cinerea, where a decrease in
fungal growth and disease symptoms on leaves was observed. On the
basis of all these findings, it can be concluded that N-isobutyl
decanamide treatment engenders enhanced resistance to necrotizing
fungi.
EXAMPLE 6
Selected Embodiments
[0029] This example describes selected embodiments of the present
disclosure, presented as a series of indexed paragraphs. In
particular, this example describes methods of using alkamides to
protect plants from pathogens.
[0030] 1. A method for using at least one member of a family of
compounds called alkamides as a defense elicitor or natural
pesticide to protect plants against the attack of pathogenic
microorganisms.
[0031] 2. A method for using at least one member of a family of
compounds called alkamides as a defense elicitor or natural
pesticide to protect plants against the attack of pathogenic fungi,
bacteria, parasites, viruses, and common plant pests.
[0032] 3. The method described in paragraph 2 or 3, where the
alkamide is N-isobutyl decanamide.
[0033] 4. The method of any of paragraphs 1-3, where one alkamide
or a combination of two or more alkamides is used as a defense
elicitor or natural pesticide, to protect plants against the attack
of pathogenic fungi, bacteria, and/or viruses.
[0034] 5. The method of any of paragraphs 1-3, where one alkamide
or a combination of two or more alkamides is used as a defense
elicitor or natural pesticide, to protect plants against the attack
of pathogenic fungi.
[0035] 6. The method of any of paragraphs 1-3, where one alkamide
or a combination of two or more alkamides is used as a defense
elicitor or natural pesticide to protect plants against the attack
of necrotrophic plant fungi.
[0036] 7. The method any of paragraphs 1-3, where one alkamide or a
combination of two or more alkamides is used as a defense elicitor
or natural pesticide to protect plants against the attack of the
pathogenic plant fungi Alternaria sp., Botrytis sp., or both.
[0037] 8. The method of paragraph 1, where the alkamide(s) is
extracted from at least one natural source by traditional solvent
extraction, supercritical solvent extraction (SFE), by organic
synthesis, or with the use of enzymes in organic solvents.
[0038] 9. The method of paragraph 1, where the alkamide(s) is
produced synthetically or partially synthetically from
precursors.
[0039] 10. The method of paragraph 1 where the alkamide(s) is used
in conjunction with other pathogen controlling agents or defense
elicitors to protect plants against the attack of pathogenic
microorganisms or other pests or parasites.
[0040] 11. The method of paragraph 1, where the alkamide(s) is
applied through one or several routes of application, such as
aerosol, direct application on plants or seedlings, and/or combined
with other nutrients or combined with fertilizers.
[0041] 12. The method of paragraph 1, where the alkamide(s) is
provided in a kit for its subsequent application in plants.
[0042] The disclosure set forth above may encompass multiple
distinct inventions with independent utility. Although each of
these inventions has been disclosed in its preferred form(s), the
specific embodiments thereof as disclosed and illustrated herein
are not to be considered in a limiting sense, because numerous
variations are possible. The subject matter of the inventions
includes all novel and nonobvious combinations and subcombinations
of the various elements, features, functions, and/or properties
disclosed herein. The following claims particularly point out
certain combinations and subcombinations regarded as novel and
nonobvious. Inventions embodied in other combinations and
subcombinations of features, functions, elements, and/or properties
may be claimed in applications claiming priority from this or a
related application. Such claims, whether directed to a different
invention or to the same invention, and whether broader, narrower,
equal, or different in scope to the original claims, also are
regarded as included within the subject matter of the inventions of
the present disclosure.
* * * * *