Note: Descriptions are shown in the official language in which they were submitted.
CA 02912714 2015-11-23
USE OF LIPO-CHITOOLIGOSACCHARIDES TO INITIATE EARLY FLOWERING
LEADING TO INCREASED FLOWER NUMBERS AND INCREASED YIELD IN
PLANTS AND RELATED METHODS AND COMPOSITIONS
Field of the Invention
The present invention relates generally to the fields of agriculture and
horticulture,
including but not limited to agricultural crops, flowers, fruits, vegetables,
nuts,
turfgrass, herbs, spices, ornamental shrubs and trees, aquatic plants, tubers,
minitubers, microtubers, corns and mushrooms grown outdoors or in greenhouses
or
indoors for both commercial or personal use and agriculture and more
specifically to
the use of Lipo-chitooligosaccharides (LCOs ) and compositions thereof to
induce
early flowering, increase the number of buds and flowers, initiate earlier
fruiting,
earlier maturity and increase yields in plants and to methods of inducing
earlier
flowering and initiation of earlier fruiting in plants by exposure to LCOs and
compositions of same.
Background of the Invention
There is a growing interest in the role of LCOs and compositions thereof for
enhancement of plant seed germination, seedling emergence and growth of plants
both for crop and horticultural purposes in both legumes and non-legumes.
Compositions for accelerating seed germination and plant growth are provided
in
Application No. PCT/CA99/00666, published February 3, 2000, WO 00/04778. There
is also an interest in the possible effects of LCOs in plant photosynthesis
and
PCT/CA00/01192, published April 19, 2001, WO 0'1/26465 Al describes the use of
LCOs and compositions of LCOs for increasing plant photosynthesis. Chemical
structures of LCOs are described in U.S. Patent Nos. 5,175,149; 5,321,011 and
5,549,718. Synthetic LCOs are also known.
There is great interest in the field of agricultural research, particularly in
the field of
plant growth promoters, of plant physiological processes which may be affected
by
LCOs. Prithiviraj et al, Planta (2003) 2'16:437-445. "A host-specific bacteria-
to-plant
signal molecule (Nod factor) enhances germination and early growth of diverse
crop
22046918.1 1
CA 02912714 2015-11-23
plants" discuss certain observed induced physiological changes in both host
and
non-host plants by LCOs. Atti et al, conference paper 'CID Young Professionals
Forum, 18th International Congress on Irrigation and Drainage, Montreal,
Canada,
2002, pages 1 to 11, discuss certain observed-psychological changes when LCOs
are administered under drought-stress conditions.
WO 01/26465 Al is directed to use of LCOs in association with increased
stomata'
conductance and enhanced photosynthetic activity. LCOs are known to be
released
by Rhizobia, symbiotic= bacteria primarily of the genera Generarhizobium,
Bradyrhizobium, Sinorhizobium, Mesorhizobium and Azorhizobium .and the like,
the
Rhizobiacese family being in a state of taxonomic flux. Both of the aforesaid
International applications summarize current understanding of the specialized
symbiotic relationship of Rhizobia with legume host plants in the formation of
nodule
organs and associated fixation of atmospheric nitrogen within these organs, as
well
as the plant to bacteria signal and bacteria to plant signal interaction
associated with
such symbiotic relationship.
Although there is a considerable body of knowledge on the influence of LCOs,
including synthetic LCOs, on typical host plant physiology, there is a growing
interest
in the effect of LCOs on plant growth with respect to both host and non-host
plants,
particularly by application of the molecule without necessarily the fostering
of a
micro-organism and plants symbiotic relationship.
The body of knowledge relating to the possible role of LCOs, including
synthetic
LCOs, in both host and non-host plants and on processes associated with plant
growth promotion continues to grow, with particular practical interest in the
effects of
LCOs on plant physiology and processes relevant to increase plant yields, not
only
with respect to commonly considered crop plants, including agricultural crops,
both
host and non-host, but also with respect to horticulture species.
Thus, there continues to be a need to study the effects of LCOs, including
synthetic
LCOs, on plant growth, in addition to processes relating to nodulation and
nitrogen
fixation in legume host plants and to germination, emergence and
photostimulation in
Z2046918.1
CA 02912714 2015-11-23
both legume and non-legume plants. In particular, there is a need to study the
effect of LCOs on bud and flowering initiation, budding, fruiting initiation
and
development, generally in relation to growth and maturity of plants, both
leguminous
and non-leguminous and the effect on plant yields, all in contrast and in
addition to
the effects as described in the above-cited references. The present invention
endeavours to address these and other needs.
Summary of the Invention
The present invention relates to the use of LCOs in initiating early flowering
and
budding, increased flowering and budding, leading to earlier fruit development
in
non-legume and legume plants, as compared to flowering and fruit development
under conditions without use of LCOs, and the enhancement of plant growth and
yield associated therewith. The present invention also relates to agricultural
compositions comprising an effective amount of at least one LCO and
agriculturally
acceptable carriers, associated with early flowering and budding, increased
flowering
and budding, earlier plant maturity and earlier initiation of fruit
development as
compared to conditions without use of LCOs, and with increased growth and
plant
yield. The present invention further relates to methods using LCOs and
compositions
of one or more LCOs and agriculturally acceptable carriers, associated with
earlier
flowering initiation and budding, increased flowering and budding and earlier
plant
maturity leading to initiation of earlier fruit development in both legume and
non-
legume crop plants as compared to conditions without use of LCOs and otherwise
associated enhancement of growth and yield, and all as exemplified herein
below.
Surprisingly, the compositions of the present invention affect not only legume
varieties but also a wide and divergent variety of non-legume plants,
including crop
plants and horticultural and bedding plant species in the initiation of
earlier flowering
and budding, increased flowering and budding, earlier maturity and earlier
fruit
development, and increased yield, as compared to conditions where LCOs are not
applied and all as exemplified herein below.
According to the present invention, in both legume and non-legume plants, the
administration of an effective amount of LCO or LCOs, or of compositions of
one or
more LCOs with agriculturally suitable carriers, initiates budding and/or
flowering at
an earlier stage, increases total bud and/or flower numbers and leading to
earlier
fruit development and plant maturity as compared to conditions without use of
LCOs,
3
22673197.1
CA 02912714 2015-11-23
including an associated increase in yield. Administration of LCOs for such
purpose
may be by leaf or stem application, or application in the proximity of the
seed, root
or plant. Such methods are non-limiting and may include other methods, which
would be understood by the skilled person, including by administration of
micro-
organisms known to release LCOs in the proximity of a plant seed, tuber, corm,
or
seedling in any stage of emergence, or in the proximity of a plant, including
in the
vicinity of the root and root hairs. The same would be with respect to
application of =
LCOs independent of the micro-organisms known to release such molecules.
Thus, in accordance with a further embodiment of the present invention, there
is
provided a method for the initiation of earlier flowering, increased budding
and
flowering, leading to earlier fruit development and plant maturity in non-
legume and
legume plants associated with the growth and yield of a plant, comprising the
treatment of a plant with an effective amount of one or more LCOs or a
composition
comprising an agriculturally effective amount of one or more LCOs in
association with
an agriculturally suitable carrier or carriers, wherein the effective amount
has the
effect of initiating earlier flowering and/or budding and/or increased bud
and/or
flower number, leading to earlier fruit development and/or plant growth and/or
yield,
as compared to an untreated plant, and all as exemplified herein below.
Suitable
LCOs for use according to the present invention include the LCOs as identified
in the
aforesaid international applications and patents.
Compositions of the present invention will be understood to include in their
scope,
one or more different LCO molecules, including synthetic molecules, as well as
comprising one or more types of molecules other than LCO, including, without
limitation, one or more plant to bacteria molecule and/or other molecules or
agents
known to promote growth or fitness and mixtures of such compositions.
The inventors and applicant herein are the first to show, as exemplified in
the
greenhouse and field experiments set out hereafter, that a composition
comprising
an LCO can have a significant effect on both legume and non-legume plants by
initiating early bud and/or flowering, increased bud and/or flowering, leading
to
earlier fruit development and/or yield, as compared to conditions without use
of
LCOs, and the enhancement of plant maturity, growth and yield associated
therewith. Non-limiting examples of crop plans include dicotyledons and
monocotyledons and legumes. From the aforesaid experiments and as set out
4
22673197.1
CA 02912714 2015-11-23
below, it can be predicted that such results will apply to crop, including
agricultural
crop, horticultural and personal use plants, legumes and non-legumes,
including, but
not limited to, flowers, fruits, vegetables, nuts, tubers, turf grass, herbs,
spices,
ornamental shrubs and trees, aquatic plants and mushrooms grown in field or
greenhouse for agricultural, commercial and personal use. In view of the
plants
exemplified herein and the results, the skilled person will appreciate, can
adapt the
teaching of the present invention to a diversity of plants, both legume and
non-
legume, for crop, horticultural and personal use, including but not limited
to, plants
of the families: Fabaceae, Brassicaceae, Solonaceae, Chenopodiaceae,
Asteraceae,
Malvaceae, Cucurbitaceae and Poaceae.
The term "LCO" as used herein, will be understood as reference in general to a
Nod
factor which is under control of at least one modulation gene common to
rhizobia,
that is bacterial strains which are involved in a nitrogen fixing symbiotic
relationship
with a legume, and which serve as micro-organism-to-plant phytohormones which
induce the formation of nodules in legumes and enable the symbiotic micro-
organisms to colorize said plant modules. LCOs,sincluding synthetic LCOs are
understood to comprise derivatives of an oligosaccharide moiety, including
fatty acid
condensed at one end thereof. Non-limiting examples of LCOs are described in
U.S.
Patent numbers 5,175,149; 5,321,011 and 5,549,718. The instant invention is
demonstrated in particular with LCOs from Bradyrhizobium japonicum, but is not
so
limited.
The uses, compositions and methods of the present invention will be understood
to
include initiation of early bud and/or flowering and/or increased flowering
and/or
budding, leading to earlier fruit development and/or enhanced plant maturity
and/or
plant growth and yield under both sub optimal or limiting and non-limiting
environmental conditions associated therewith. Such sub optimal or limiting
environmental conditions include but are not limited to liming or sub optimal
conditions of heat, water pH, soil nitrogen concentrations and the like.
An effective amount of LCO will be understood to relate to uses, compositions
and
methods of the present invention wherein the amount is sufficient to manifest
statistically significant earlier budding and/or flowering and/or increased
flowering
and/or budding, leading to earlier fruit development and/or enhanced maturity
and/or plant growth and yield associated therewith.
=
22673197.1
CA 02912714 2015-11-23
By proximity of seed, tuber, corm, root or plant will be understood to relate
to any
location of seed, root or plant wherein soluble materials or compositions of
the
present invention will be in actual contact with said seed, root or plant.
By bud or budding will be understood conditions consistent with stem swelling
consisting of overlapping immature leaves or petals. By flowering will be
understood
the process or state of producing one or more flower.
5A
22673197.1
CA 02912714 2015-11-23
Brief Description of fhe Drawings
The invention having been generally described above, the accompanying figures
will
novi be referenced In the disoussion of a preferred embodiment of the
invention, as
set out in the examples which follow, in which:
Figure 1 shows the effect of LCO dose and timing on fruit set of Cobra
tomatoes;
(Same data as tabie 2)
Figure 2 shows the effect of LCO dose and timing on fruit number of Cobra
tomatoes;
(Same data as table 2)
Figure 3 shows the effect of LCO dose and timing on flower number of Cobra
tomatoes; (Same data as table 1)
Figure 4 shows the effect of LCO dose and timing on flower number of Cobra
tomatoes; (Same data as table 1)
Figure 5 shows the effect of LCO dose on number of flower of Cobra tomatoes;
(Same data as table 3)
Figbre 6 shows the effect of LCO dose on the number of fruit of Cobra
tomatoes;
(Same data as table 4)
Figure 7 shovvs the effect of LCO dose on yield of fruit a Cobra tomatoes;
(Same
data as table 5)
Figure 8 shows the effect of LCO on tomato plant flowering;
Figure 9 shows the effect of LCO on induction of flowering in Arabidapsis
thaliana;
Figure ld shows the effect of LCO on induction of flowering in Arabidopsis
thaliana;
Figure 11 shows the effect of LCO dose on thè yield of fruit per plant,
in.tomato plant
application; and (Combined with figure 13)
Fig. 2-1*. LCO foliar application enhanced early flowering and total flower
number in
greenhouse tomatoes.
Fig. 2-2; LCO foliar application = enhanced early fruiting and total fruit
number in
greenhouse tomatoes.
-6-
=
CA 02912714 2015-11-23
Fig. 2.3: Effect of LCO application on earlier flowering and number of flowers
in
Marigolds.
F10. 2.4: Effect of LCO application on fruit number of strawberries.
Fig. 2.5: Effect of LCO sail application on cherry tomato early fruit numbers.
Fig. 2.6: LCO application promoted tomato early fruit number.
Fig. 2.7: LCO application promoted tomato early fruit.
Fig. 2.8: Cumulative harvested fruit number from tomato plants when 5Ong/plant
LVO was applied once at variable growing stages.
Fig. 2.9: Cumulative harvested fruit yield from tomato plants when 5Ong/plant
LCO
was applied once at variable growing stages.
Fig. 2.10: Effect of LCO application on advancement of hot pepper early
flowering.
Fig. 2.11: Effect of LCO application on advancement of hot pepper fruiting.
Other objects, advantages and features of the present invention will become
more
apparent upon reading of the following description of preferred embodiments,
which
is non-restrictive, and with reference to the accompanying figures, which is
exemplary and should not be interpreted as limiting the scope of the present
invention,
Description of the Preferred Embodiment
The following experiments are reported herein, conducted to study the effect
of
application of LCO on the initiation of flowering and fruit development of a
host plant
under both greenhouse and field conditions.
Trials 1 and 2: Effect of LCO on Greenhouse Tomato
Two experiments on the application of LCO to Tomatoes have been undertaken
Trial Cobra (a hybrid
cultivar) was used to examine optimum application dose
between 10 and 100 ngiplant at one or two applications and the LCO delivery
medium. The levels chosen were the extremes of beneficial doses deten-nined
previously in tomato field trials. The delivery media tested were LCO in
water, Apex
-7-
CA 02912714 2015-11-23
and centrifuged Apex. The first application of LCO was made 10 days after
transplanting. When applied a second time it was 2 weeks after the first
application.
The following parameters were tested: leaf number, plant height, number of
cluster,
number of flower, number of fruit. Observations were made continuously for
approximately a two-month period, at one-week intervals until plant growth was
limited by pot bound roots.
A statistically significant difference in early yield was noted between 5Ong
LCO
treatment applied twice and the untreated control (see Table 5, Figure 7,
Fruit
weight). Other levels of application were not significantly better in yield
than contol.
There was statistically significant effect between 5Ong LCO treatment and
control on
fruit weight of Cobra. 5Ong treatment showed more uniform results in different
delivery media. The increase of early yield by 5Ong LCO showed the potential
ability
of LCO applied as a growth enhancer on tomatoes.
There was no statistical difference between treatments on flower number and
number of fruit set at any time point. However, the double application of 5Ong
LCO
per plant provided a numerically higher flower number earlier and also
delivered the
best early yield. Similarly, There was no significant difference on number of
fruit
among LCO treatments. Fruits appeared 48 days after transplanting and 38 days
after first application of treatments and 24 days after second application of
treatments. Plants in LCO 10 rig treatment showed slightly higher number of
fruit
than other treatments and than control.
Examination of the first graph in Trial 'I (Figure 5, Table 3) indicates a 4-5
day
advance of flowering over control and the second graph demonstrates an 8-9 day
advance in early fruiting over control (horizontal separation between
treatment lines).
Early fruiting must arise from earlier flowering.
There was no significant difference on number of flowers among LCO treatments.
Flower buds appeared 30 days after transplanting and 20 days after first
application
of treatments. Flowers started to open 40 days after transplanting, and 30
days after
first appiication of treatments. The plants in 50 ng LCO treatment had more
flowers
than other treatments and control at 21% and 14% on Jan. 15 and Jan 22. On the
other days, the number of flower was similar among treatments and control.
-8-
CA 02912714 2015-11-23
Trial 2: The Cobra variety was used to re-examine optimal doses of LCO. The
concentrations tested were 5Ong and 75ng LCO per plant, applied once (2 weeks
after transplanting) and twice (4 weeks after transplanting). ,The sample
number was
increased to 20 plants.
At 50 ng/plant there was a significant difference from control on number of
flowers
over the first three observations (Table 1) (see Dose & timing on Flower No.
Cobra
Trial 2). Later, treatment significance on flowering was lost but this is to
be expected
because of the flowering characteristics of the tomato plant. Examination of
the
Flower number data for Cobra Trial 2, (Figures 3 and 4) indicates an advance
in
flowering of some 3 days for 50 ng treatment attaining same flower number as
control. On fruit number the 5Ong LCO treatment applied twice showed
significantly
higher numbers over control for the first 4 weeks. The higher fruit number
(Table 2)
(see histogram for Fruit number Cobra Trial 2) arises from earlier flowering.
The
graph for Dose and Timing on Fruit Set Cobra Trial 2 (Figures 1 and 2)
demonstrates
that a 50 ng/plant application twice, advances equivalent fruit numbers by 2
weeks
over control.
Table 1: The Effect of Different LCO Concentrations on Flower Number of Cobra
Tomatoes in Greenhouse Studies
Treatments Flower j Flower Flower Flower Flower Flower
Number/ Number/ Number/ Number/ Number/ Number/
Plant Plant Plant Plant Plant Plant
(Feb.28) (Mar.7) (Mar.14) (Mar.21) (Mar.28) (April 4)
LCO 5Ong once 0.0 b 2.5 ab 7.4 ab 14.55 23.95 32.45
LCO 5Ong twice 0.2 a 3.2 a 8.4 a '16.0 = 25.15 =
32.2
LCO 75ng once 0.0 b 2.85 ab 7.35 ab 14.2 21.4 28.0
LCO 75ng twice 0.0 b 3.25 a 7.35 ab '15.4 23.35 31.0
Surfactant 0.0 b 1.9 b 5.8 b 12.85 21 .65 32.0
500ppm once
Significance* P=0.0006 P=0.03 P=0.05 P=0.168 P=0.156 P=0.368
* There is significant different when P<0.05
Notes; Seeding: Jan.6, Transplanting: Feb.7, First Application: Feb.21, Second
Application: Mar. 7, 2003 (Greenhouse Tomato Cobra)
-9-
CA 02912714 2015-11-23
Table 2: The Effect of Different LCO Concentrations on Fruit Number of Cobra
Tomatoes in Greenhouse Studies
-Treatments Fruit Fruit I Fruit Fruit
Number/ Number/ Number/ Number/
Plant Plant Plant Plant
(Mar.141 plar.21) (Mar.28) (April 4)
LCO 50nd once 0.05 b 0.25 d 0.525 c 1.3 b
LCO 5Ong twice -1 0.75 a 1.8 a I 2.025 a 2.5 a
LCO 75ng once 0.45 ab 0.9 bc 1.15 bc '1.45 b
LCO 75ng twice 0.6 ab ' 1.0 b 1.325 ab 1.9 ab
Surfactant 500ppm 0.1 b 0.4 cd 0.7 bc 1.4 b
once
Significance* P=0.03 P<0.0001 P<0.0001 P=0.01
' There is signtficant different when P<0.05
Notes: Seeding: Jan.6, Transplanting: Feb.7, First Application: Feb.21, Second
Application: Mar. 7, 2003.
Results: Effect of LCO Dose on Greenhouse Tomato (Cobra)
The cobra seedlings were transplanted 32 days after seeding, the first
application
was 10 days thereafter, the second application was 14 days after the first
application.
The fruits were harvested 6 weeks after the second application.
Results: Effect Of LCO Dose on Greenhouse Tomato (Cobra)
Table 3. Number of Flowers/Plant
Date
Treatment Dec. 30 Jan. 07 Jan. 15 Jan. 22 Jan. 31
Control 0.7 2 4.2 5.8 8.9
lOng 1.2 2A 4.4 5.6 8.1
5Ong 0.97 2.5 5.2 6.7 8.7
10Ong 0.9 2.2 4A 6.2 8.7
IMean 0.9425 2.275 4.55 , 6.075 8.6
-10-
CA 02912714 2015-11-23
There was no significant difference on number of flowers among LCO treatments.
Flower buds appeared 30 days after transplanting and 20 days after first
application
of treatments. Flowers started to open 40 days after transplanting, and 30
days after
the first application of treatments. The plants with a 50 ng LCO treatment had
more
flowers than other treatments and control at 21% and 14% on Jan. 15 and Jan
22.
Table 4. Number of Fruit/Plant
Treatments Date
Jan. 07 1 Jan. 15 Jan. 22 Jan. 31
Control 0.1 1.6 2 2.7
lOng I 0.3 1.8 2.1 2.5
" ____________________________________________
5Ong 0.33 2.2 2.5 2.9
1-10Ong 0.37 I 1.7 2.1 2.6
Mean I 0.275 1.825 2.175 I 2,675
Notes; See Table 3 above.
LCO application at all levels advanced early fruit set, three to four weeks
after ,
= application. The optimal application was approximately 50ng per plant.
Table 5. Fruit Weight (Gram/Plant)
Base Treatments
Ong lOng 5Ong 10Ong
Water 92.81 l 96.11 108.99 88.57
Bacterial Carrier 74.14 97.97 103.32 I 66.26 =
Bacterial Supernatentõ 61.65 67.2 '100.48 109,13
Mean 76.2 87.09 104.26 87.99
Notes; See Table 3 above.
There was statistically significant effect between 5Ong LCO treatment and
control on
fruit weight of Cobra. 5Ong treatment showed more uniform result in different
delivery
-11-
CA 02912714 2015-11-23
medium. The increase of early yield by 50ng LCO showed the potential ability
of LCO
applied as a growth enhancer on tomatoes. Water was the optimal carrier for
LCO
application in this study.
Experiments 1 and 2
Summary Of The Experiment: Effect Of LCO On Tomato Flowering
Two experiments were performed to investigate effect of LCO on plant's
flowering
using greenhouse-grown tomato plants.
In general, LCO induced early flowering in both experiments (Figures 8 and 9).
Experiment 1: LCO treatment induced flowering in 25% more plants as compared
to
control at day 1 of assessment. This increase was sustained thorough out
experiment, reaching 35% difference at day 4 of assessment. LCO treatment
caused
a 3 day-shift in time of flowering, i.e., over 80% of LCO treated plants
flowered 3
clays earlier than control, non-treated plants. Early flowering will initiate
earlier fruit
= set and subsequent earlier fruit development, which in turn leads to
higher yield of
tomatoes.
Experiment 2: initial assessment of tomato flowering confirmed again that LCO
= treatment induces early flowering in tomatoes. Initially, there is a 10%
difference
between LCO treated plants and control. This difference increased to 20% by
day 3
of assessment. Initial data obtained in this experiment confirms findings from
previous one and further supports claim that LCO treatment incuses early
flowering
in plants.
Experiments 3 and 4
Summary of the experiment: Effect of LCO on flowering in
Arabidopsis thaliana
Two experiments were performed to investigate effect of LCO on plant's
flowering
using experimental model plant Arabidopsis thaliana.
In general, LCO induced early flowering in both experiments (Figures 10 and
11).
Plants were treated with various a range of LCO concentrations. It was found
that
treatment with 10-7 Molar the most effective in induction of flowering. The
LCO
-12-
CA 02912714 2015-11-23
treated plants reached over 80% of plants with open flowers 4 days earlier
than
control, surfactant treated plants. LCO induced faster and more uniform
flowering.
Experiment 5: Foliar Application Of LCO To Bedding Plants
This is a growth room study. Seeds of garden plant species were selected on
the
basis of seed-purchase popularity (Norseco, Montreal), grown in trays of Fro-
Mix
(NB. trademarked name) Seeding Medium, and at some size were transplanted into
trays of 36 and 32 wells containing the same medium. Growth proceeded under
lights in the growth room.
Some 2 weeks before expected flowering, 16 young plants were sprayed with each
of various levels of LCO leaving control plants untreated. The results are
presented
in Table 5A on bud formation and where possible opened flowers.
Table 5A Effect of LCO Application on ornamental plants
Treatments Total Number of Impatiens Total Number of Marigold
buds buds
Untreated Control 68 26 buds and 1 flower
20 m1/16 plants of 10-7 M 71 26 buds and 0 flower
LCO application
50 m1/16 plants of 3x10-8 66 34 buds and 3 flowers
M LCO application
20 m1/16 plants of 10-8 M 85 , 24 buds and 1 flower
LCO application
20 m1/16 plants of 10-9 M 65 25 buds and 2 flower
LCO application
lt was also noted that with Marigolds all the LCO treatments produced some
plants
with 3 flower buds and the 50 ng treatment had some (2) with 4 flower buds per
plant. No untreated control plant had more than 2 buds per plant.
Trial 3: Flowering And Yield Benefit From Foliar Application Of LCO
Summer Field Trial at Macdonald College Research Farm
An investigation was conducted to examine whether foliar applications of LCO
led to
increased yield in tomato crop production. To determine concentrations or
strengths
-13-
CA 02912714 2015-11-23
to be applicable, testing was conducted with a logarithmic increase in
strength from 1
nanogram (ng) to 1000 ng/plant sprayed once, and on half, twice.
The trial results are presented in the following Figure 12 and Tables 6 and 7.
The
parameter of interest was ripened fruit which was harvested 2 or 3 times a
week,
recording each time, both fruit weight and number of fruit per set of
replicates. It was
known that fruit arise from pollinated flowers and that an increase in the one
leads to
the other. Figure 12 records cumulative harvested (red) fruit per treatment.
For the
single LCO application it will be se.en that the 10 and 100 ng/plant
treatments have
advanced fruiting by some 10 days over control (horizontal separation in
weeks).
That advance has allowed the plant to bear and ripen more fruit over the
season for
these treatments (see Figure 12 note height over control and Tables 6 and 7
for
actual weights and numbers harvested). Table 6 records harvested weight and
numbers of ripened fruit over season and it can be seen that the average
weight of
the tomatoes is not different between treatments and control. Thus the
increase in
harvested weight was due to an increase in numbers harvested, in agreement
with
actual enumeration. Table 7 demonstrates that the yield increase over the
season
was a statistically significant 17% for single application of 10 ng LCO/plant
and
agrees with Table 6 where numbers for this application were similarly
increased -
some 20%.
= Fruit numbers in treated plants are increased by 17%-20% arising from a
similar
increase in numbers of flowers able to be pollinated.
From Figure 12 there is a shift to earlier flowering when plants are treated
with LCOs
at specific concentrations, the concentrations required for physiological
change being
typical of a phytohormone where a very narrow range of concentration at very
low
concentrations is of benefit - higher and lower concentrations have no effect.
Second applications are similar in trend but less clear in analysis because
the
second application led to a later increase in unharvestable green fruit
stopped from
ripening by impending frost. This would not be a problem in greenhouse
operations
where this indetem-linate plant continues to yield over many months as long as
root
fed.
-14-
CA 02912714 2015-11-23
Table B. LCO on Tomatoes
Yield in Kg and Numbers
Harvest I Cumulative Yield (kg) to - Total No. of Tomatoes ' Average Wt of
Tomato Increase in ¨
Sept 23. per treatment in Gms No. a
No. of Applicationsl Total weight for 24 plants Tomatoes
-LCO Applied LCO 'Applied -LCO Applied r LCO Applied LCO Applied LCO
Applied from 1 to 2
ng LCO per plant _ once Twice once Twice _ once Twice
applications
¨
1 57.7200 63.8760 300 321 0.192 0,199 21
72-2240 74.1720 363 374 0.199 0.193 11
100 55.4720 67.0000 351 343 0.187 0.195 -8
1000 62.6760 71.0120 326 361 1 0.192 0.197 35
control-water 61.3840 65.8880 309 328 0,199 0.201
19
i -
[ Total No. of tomatoes produced using I
1 , _____________
Total No. of tomatoes produced
I single application of LCO at various using double
application of LCO at .
levels '
= various levels
.
1
70 _____________________________ 390 ___________________
I
1 350 _____________________
I ., = 370 . ________________
I a a
1 0330 - ti 350 i _________________
E 330 1 _______________________________________
,
1 0310 ____________________ ' o l
IP¨ . 310
. t
I 466 290 = = .
I z 271 ill 111 a IIII I e
250 1 1111 1111 1111 NI 11 270
___________________________________________________________ I
0 I 1 I 10 100 1000 0 I 1 i 10 r ion
woo i
326 328 i 321 i 374 i 343
ng LCO applied per ng LCO apptied par ptant
COnatSion3
'
1 Treatments did not atter size of tomatoes
2 Treatments determined the number of tomatoes and therefore yield
3 Ail doses over 10 ng LCO per plant increased yield total number
4 Between 10 and 100 ng appears to be the best application rate
5 Double application did not improve yields significantly over a ,single
application
-15-
CA 02912714 2015-11-23
Table 7 LCO on Tomatoes
Cumulative yield data to Sept 23
Each data point is the average of 4 randomized rows, each of 6 plants
Yield ir Kg per plant
Harvest Cumulative Harvest I Yield increase Yield I Percent Yield
to Sept 23. over Control Increase ' inc. over Control
No. of Applications Applied Applied Applied Applied for 2 no'
Applied Applied
once Twice once Twice Application once Twice
(24 plant (24 plant
ng LCO per plant Average) Average)
1 Not as good as Control , 0.0000 ¨ 0.00 --
3.0093 3.0905 0A517 0.3452 0.48 17.7% 12.6%
100 2.7280 2.7917 0.1703 0.0463
0.06 6,7% 1.7%
1000 2.6115 2.9588 = 0.0538 0.2135 0.35 2.1% 7.8%
control -water 2.5577 2_7453 ¨ ¨ 0.19
Yield per plant over control from single Yiekt response due to 2nd
application
application of LCO at various levels of LCO
(1 to 1000 ngtplant)
0.5000 ___________________ 0.40 ________________
0A000 __________________ t. 0.30 .
03090 ________________________________________ 1
?.4
9 020.2000 = ;
-0
ir-
=
1
. OCCO ___________________ MOO _________________
=
io I oo ioao, i io loo j low
0 0.451666710.1703333 O0538333I ¨ 0 i0.08116667
0.063556670.347322331
=
=
-16-
CA 02912714 2015-11-23
Experiment 6: Foliar Application of LCO at Variable Growth Stages
Table 2-1 Response Of Tomato Fruit Yield To LCO Foliar Application At Variable
Growth Stages
Weight Average % of .
Fruit on (kg) per 24 Weight Fruit # % weight vs.
LCO Applied Time 24 plants plants (glfruit) vs.
control control
DATP* (7/4) 1276ab 15188ab 118.2 28.63 10.8
DATP (7/15) 1260ab 163.78ab 130.0 27.02 20.3
DATP (7/25) 1199abc 160.11ab 133.5 20.87 17.6
DATP (8/8) 1318ab 155.89ab 118.3 32.86 14.5
DATP (8/18) 1115bc 154.62ab 138.7 12.40 13.6
10+20 DATP (7/4+7/15) 1125bc 153.43ab 136.4 13.41 12.7
10+20+30 DATP
(7/04+7/15+7/25) 1282ab 161.89ab 126.3 29.23
18.9
10+20+30+40 DATP
(7/4+7/15+7/25+8r7) 1191abc 152.50ab 128.0 20.06
12.0
10+20+30+40+50 DATP
(7/4+7/15+7/25+7/8+8/1
8) 1373a 174.07a 126.8 38.41 27.8
Untreated control (UC) 992c 136.15b 137.3 0 0
Significance at 5% Yes Yes No No No
DATP,standards for Days After Transplanting
Tomato seedlings (6-leaf stage) were transplanted in farmland. The plants were
watered on the day that they were transplanted and whenever the soil was very
dry
during the season. Fertilizer (20-20-20) was applied at 250kg/ha to the tomato
field
before transplantation. All tomato plants were supported by sticks when they
were
heavily loaded with fruit. Fruit yield (in the table) was finally cumulated at
the end of
the season.
Compared to the untreated control, LCO increased fruit number (up to 38.4%)
and
total fruit weight (up to 27.8%). Five of 9 treatments had significantly
increased fruit
number over control. For a single application of LCO, the best time is 20-40
days
-17-
CA 02912714 2015-11-23
after transplantation. Multiple applications led to increased fruit yields
over a single
application but these results were not significantly significant.
Experiment 7: Foliar Application of LCO on Pepper
Table 2-2 Effect Of LCO Foliar Application On Pepper Early Fruit Maturity
(Sept 17)
And Final Yield (Oct 9) In Horticulture Centre 2003.
Treatments First 4wk yield
Fruit/plant gram/plant
14 ng/plant 5.79ab 784.91ab
=
50 ng/plant 5.83ab 833.79ab
75 ng/plant 5.77ab 835.26ab
100 ng/plant 4.87b 694.48b
141 ng/plant 6.48a 934.83a
282 ng/plant 5.02ab 702.87b
Water 4.87b 643.37b
Significant at 5% Yes Yes
13 pepper seedlings (cv. Camelot, 6-leaf stage) were transplanted in 2 rows
per plot
(3.5x2.5M2). The rows were covered with 65cm width black plastic mulch one
week
before transplanting. Fertilizer (10-52-10) solution of 250ppm was applied
into the
planting hole through the mulch when transplanting (approx. 250m1 per plant).
The =
drip irrigation system was set to twice a week and 4 hours each time,
depending on
the soil moisture. Plants were sprayed with LCO 14 days after transplanting
(5m1/plant) and 27 days after transplanting (50m1iplant).
LCO foliar application significantly increased fruit number in the early
stages by
approximately 1 fruit per plant. Treatment of 141ng/plant (5m1 of 2 x 104M)
was the
best dose.
=
-1 5-
=
CA 02912714 2015-11-23
Experiment 8: Foliar Application of LCO on Corn
Table 2-3. Effect Of Foliar Application Of LCO On Sweet Corn Ear Number
Treatments Ears/ha Ear Marketable Average Ear
Weight ears/ha Length(cm)/ear No/plant
(kg) /ha
10:7 M once 65416.7 ab 4383.3 25004.0 13.'12 e 0.934
104 M once 67083.3 ab 4800.0 26250.0 '13.24 bc 0.905
10-9 M once 68333.3 a 4266.7 29166.7 '13.68 abc 0.928
10' M twice 70833.3 a 5000.0 30416.7 13.8'1 ab 0.935
104 [VI twice 62083.3 ab 5150.0 30000.0 '13.64 abc 0.856
10' M twice 56666.7 be 4033.3 25000.0 13.43 bc 0.886
Water 49166.7 c 4416.7 39583.3 14.23 a 0.760 =
Control
Significance P<0.05 NS NS P<0.05 NS
Fertilizer (36-12-18) was applied to the corn field at 500kg/ha before
seeding. A
machine planter was employed to sow sweet corn grain. Plot size was 4x4.5=18
M2,
and 6 rows/plot. To protect corn ears from raccoons and other animal damage,
sweet corn plants were protected with an electric fence around the plots after
silking.
LCO was applied once 40 days after sowing or/and twice, 40 days after sowing
and
58 days after sowing at a rate of 200L/ha for the first application and
300L/ha for the
second application. Com was harvested 80 days after sowing, from the two
middle
rows and corn ears reaching 12cm or longer were counted as marketable.
Foliar application of LCO in the range of 104- 104M significantly increased
total
number of sweet corn ears. Total ear weight and marketable ear number were not
increased by the treatments in these experiments as it was necessary to
harvest the
crop before ail were fully ripened.
-19-
CA 02912714 2015-11-23
Experiment 9: Effect of LCO on Grain Com Yield
Table 2-4. Effect of foliar application of LCO on the fresh and dry yield of
grain corn
Treatments Fresh Yield Fresh Yield Dry Yield Dry yield Ears/ 2
(kg/2 rows) (kg/ha) (kg/2 rows) (kg/ha) rows
10-7 M once 4.40 7333.3 3.44 5730.6 37.25ab
104 M once 4.57 7616.7 3.62 6040.4 37.00ab
10-9 M once 4.75 7916.7 3.77 6275.6 39.00ab
104 M twice 5.03 8383.3 3.96 6606.5 40.00a
104 M twice 4.12 6866.7 3.23 5387.1 32.25bc
10-9 M twice 4,55 7583.3 3.57 5953.5 37.25ab
Water Control 4.26 7100.0 3.38 5637.6 32.00c
Significance NS NS NS NS P<0.05
Fertilizer (36-12-18) was applied to the corn field at a rate of 500kg/ha
before
seeding. A machine planter was employed to plant the grain (cv. DK376, 1-
IL12650,
= Fludioxnil coated). To protect corn ears from bird damage, grain corn
ears in the
middle two rows were covered with plastic nets after silking. LCO was applied
to
, corn plants at a rate of 200Uha for the first application 40 days after
planting and at
400Uha for the second application 58 days after planting. The two protected
middle
rows of plants were harvested by a combine 152 days after planting. Ear number
was significantly" increased by all treatrnents of LCO application over
untreated
control, except for the 10-8M double application. The total grain yield
increased for all
but the 10-8M double application.
-20-
CA 02912714 2015-11-23
Experiment 10: Effect of LCO on Ridgetwon Tomato
Table 2-5. Effect Of LCO Application On Ridgetwon Canning Tomato (The First
Harvesting Data)
Treatments Fruit number Increase vs. Fruit weight Increase
vs.
(on 24 plants) CK (%) (kg/24 plants) CK (%)
50-0-0 206AB 12.6% 11.916 9.9%
0-50-0 209AB 14.2% 12.598 16.2%
50-50-0 219A 19.7% 12.567
50-75-0 199AB 8.7% 11.421 5.3%
0-50-75 191A8 4.4% 11.352 4.7%
Control 183B 0 10.844 0
Significant at Yes No
5%
The experiment was conducted at Ridgetwon College, University of Guelph,
Ridgetown, Ontario. Tomatoes were transplanted in single twin rows, 7m in
length
spaced 1.65m apart. Treatments of LCO were applied three times, two weeks
before
flowering (28 days after transplant), two weeks after flowering (52 days after
transplant) and six weeks after flowering (69 days after transplant). Spray
applications were applied using a specialized, small plot research CO2 sprayer
with a
two-nozzled, hand-held boom applying 2001.1ha of spray. Rates were determined
based on 38 tomato plants per plot, replicated 4 times, equaling 152 plants
per
treatment. Early fruit was harvested for yield evaluation on August 20,2003.
LCO
foliar spray applied to tomato plants at 2 weeks before and after flowering
significantly increased fruit number by up to 20% and also increased fruit
weight by
up to 16%.
Experiment 11: LCO Foliar Application in Greenhouse Tomatoes
Tomatoes were seeded and transplanted into 10" pots 30 days later in the
greenhouse. Plants were sprayed with 5 ml (50ng) LCO solution per plant 10
days
after transplant and 14 days after transplant (50 ng x2). Flowering data was
collected
28 days after transplant.
LCO improved tomato early flowering, and a 50 ng/plant single application
better
than a double application. All applications were better than control. See
Figure 2-1.
The same plants as fig. 2-1 were sampled for fruit data 28 days after
transplant.
-21-
CA 02912714 2015-11-23
There were no fruit seen on the control plants at this moment, however, foliar
application of LCO increased tomato early fruit setting under greenhouse
conditions.
Treatment of a single 50 ng LCO application increased fruit set by approx. 1
fruit/plant. See Figure 2-2.
Experiment 12: LCO Application on Marigolds
Marigolds were planted in 32-cell flat and LCO was applied foliarly to plants
4 weeks
after sowing (4 flats/treatment, 1 rnitplant applied containing various levels
of LCO).
Data collection started from the first flower appearing.
The higher doses of LCO (100-200 ng/plant) enhanced flowering in the first 2
weeks
after application, whereas the lower doses (10-50 ng/plant) showed better
enhancement of flower 3 weeks after application. The best treatments advanced
flowering by 2 days and the number of flowers at 25 days by 8%, See Figure 2-
3.
Experirnent 13: LCO Application on Strawberries
Field strawberries were sprayed with a foliar application of LCO at three
dosages on
the= same day, as set out in Fig. 2-4. Fruit was harvested 2-3 times a week,
beginning 24 days after application.
= Treatment of LCO at 10-8 M (70ng/ plant) increased early fruit setting
and fruit
number 3-7 weeks after application from 7 to 30%.
Experiment 14: LCO Application on Cherry Tomatoes
Cherry tomato seedlings (5-week oid) were transplanted into 5" pots in the
greenhouse. LCO solutions were prepared with water and 50m1/plant was applied
to
the soil in the pot after transplantation. Ripened fruit (orange or red) were
collected 8
Weeks after transplantation.
LCO soil applied to transplanted cherry tomato enhanced early fruit number.
LCO 10
ng per plant by soil application showed the best fruit enhancement at the
early stage.
See Figure 2-5.
-22-
CA 02912714 2015-11-23
Experiment 15: LCO Application on Early Fruit Number and Yield
Red tomato seedlings (cv. Mountain Spring) were transplanted at their 4-leaf
stage.
7 plants in one row were transplanted in each plot of 3.5x 2.5M2. The row was
covered with 65cm width black plastic mulch, one week before transplanting.
Fertilizer (10-52-10) solution of 250ppm was applied into the planting hole
through
the mulch when transplanting (approx. 250m1/plant). The drip irrigation system
was
set to twice a week and 4 hours each time, depending on the soil moisture.
Plants
were sprayed with LCO 15 days after transplant (5m1/plant) and 29 days after
transplant (20m1/plant). Fruits were first harvested 67 days after transplant.
LCO application significantly increased early fruit number and weight, but did
not
increase the average fruit size. The optimal application was 75ng/plant. See
Figures
2-6 and 2-7.
Experiment 16: LCO Application on Fruit Number and Weight at End of Season
Tomato seedlings (6 leaf-stage) were transplanted. The plants were watered on
the
day they were transplanted and whenever the soil was very dry during the
season.
Fertilizer (20-20-20) was applied at 250kg/ha to the tomato field before
transplantation. Al! tomato plants were supported with sticks when they were
heavily
loaded with fruit. Fruit yield was finally cumulated at the end of the season
115 days
after transplant.
Data showed the optimal application was 20-40 days after transplantation.
During
this period, LCO applied once at 5Ong increased fruit number by up to 33% and
fruit
weight by up to %. See Figures 2-8 and 2-9.
Experiment 17: Effect of LCO on Hot Pepper Flowering and Fruiting
30-day old seedlings were transplanted into 5" pots and 20 days later (20 DAT)
plants received the first LCO spray at 2m1/plant (50ng/plant). The 2nd spray
was
conducted 3 weeks (41 DAT) after the first. Data was collected 5 weeks (55
DAT)
after the first LCO application.
LCO applied in single or double applications increased early flowers up to 5%
and
40% over control, respectively at 5 weeks. See Figures 2-10 and 2-11.
-23-
CA 02912714 2015-11-23
30-day old seedlings were transplanted into 5" pots and 20 days later (20 DAT)
plants received the first LCO spray at 2m1/plant (50ng/plant). The 2nd spray
was
conducted 3 weeks (41 DAT) after the first. Data was collected 5 weeks (55
DAT)
after the first LCO application.
LCO applied in single or double applications increased the number of early
fruits by
up to 159% and 284% over control, respectively, in 5 weeks. See Figure 2-11.
Experiment 18: LCO Application on Legume
Table 8. Effect of LCO foliar application on grain yield and biomass of
Legumes
Treatments = Biomass (g/5-plant) Yield (kg/ha)
Applied once Applied twice Applied once Applied twice
LCO 1 ng 49.46 AB 55.10 AB 2563.96 AB 2673.54 AB
1.LCO lOng 46.28 AB 60.11 A = 2515.33 B = 2385.75 B
LCO 10Ong 46.87 AB I 55.09 AB 2635.00 AB 2974.17 A
LCO 1000ng = 47.99 AB I 47.81 AB 2452.71 B 2620.42 AB
Water 45.07 B 55.13 AB 2293.25 B 2421.88 B
Untreated 1.1.1 N/A 1.1.2 2285.33 13
control
A short heat-unit variety of soybean (cv. Nortman, HU 2425) was planted at
density of plants on the field of approx. 300 plants per plot (500,000
plants/ha). The soybean plants were first treated with LCO at their blooming
stage 24 days after planting. The treatment amounts of LCO (detailed above)
were diluted with distilled water to give a 2-litre solution sprayed over 4
plots
of the treatment. The second application was sprayed at the podding stage
49 days after planting. As before, the LCO was diluted with distilled water to
2-litre solution per 4 plots of the treatment. The plants receiving a single
application were sprayed with LCO for the first time, whereas the plants
receiving a double application were sprayed for the second time. A CO2
pressure sprayer was employed for this trial. The amount of fluid dispensed
-24-
CA 02912714 2015-11-23
by the sprayer was controlled by the nozzle size. It was calibrated with water
prior to spraying with LCO. Biomass was examined 58 days after planting by
.digging out by hand 5 plants per plot. The final yield was obtained by
harvesting by combine the intact area (2-meter long to the end) 101 days'after
planting. The data were analyzed with the SAS program.
Although the present invention has been described hereinabove by way of
preferred
embodiments thereof, it can be modified. The scope of the claims should not be
limited
by the preferred embodiments set forth in the examples but should be given the
broadest
interpretation consistent with the description as a whole.
-25-
