Note: Descriptions are shown in the official language in which they were submitted.
WO90/13Z24 PCT/US90/02240
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METHOD OF ENDOPHYTE-ENHANCED PROTECTION OF PLANTS
FIELD OF THE INVENTION
The present invention relates to a method of
providing enhanced protection agai.nst disease in
commercially-valuable plants. More particularly, the
present invention relates to such a method employing
endophytic microorganism~.
BAC~GROUND OF TXE INVENTION
Since the first development of agriculture, man has
battled to protect ~aluable plants from attack by bacteria,
viruses, fungi, and insect pests that can rob him of the
product of his labor and, on occasion, even thxeaten his
existence. The focus in the past has been on chemical
means of protection. Recently, the increased awareness of
~he effects of chemicals on the environment has led to the
search for other, less toxic means of protecting plants.
Mechanisms for biological control may provide a solution to
this problem; however, to date they have proven to be
largely ineffective.
Biological control of plant pathogens can be
defined as "the decrease of inoculum or the disease-
producing activity of a pathogen accomplishe~ through one
or more organisms, including the host plant but excluding
man.~' See, K.F. Baker, Annual Review of Phytopathology 28:
67-8S (1987). The term was first used in relation to plant
pathogens in 1914 and to insects in 1919.
One form of biological control is the phenomenon of
induced resistance, that is, an increase in a plant's
ability to resist disease after prior exposure to a
pathogen. Although the mechanism of action of induced
resistance (also called cross protection, acquired
resistance or acquired immunity) has never been fully
understood, it has been reported in the scientific
literature since the 1950's. C.W. Bennett described virus
infection to protect plants by induced resistance in
Advance~ in Virus Research 1:39 ~1953). Other early
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reports on induced resistance include T.O. Biener (Annual
Review of Phytopathology 1:197 (1963)) and B. Kassanis
(Advances in Virus Research 10:219 (1963)).
These early reports of induced resistance described
resistance that was conferred to plants by the introduction
of biotic inducers, i.e., pathogenic inducers that were
either live or attenuated (i.e., unable to live and
increase within the plant). Although these inducers may
have created the desired response in the plant, target
crops and non-target species were subjected to pathogens
that could be potentially harmful to them. In addition,
these pathogens were often applied topically thus enhancing
the opportunities for environmentally mediated inactivation
of the organism (e.g. W degradation). Moreoverj topical
application required relatively large amounts of the
pathogen, enhancing the opportunity for unwanted exposure
of non-target species. In addition when attenuated
pathogens were us~d, multiple applications were often
required.
In addition to the biotic inducers described above,
abiotic (i.e., biochemical) inducers have also been
reported in the scientific literature, (~odderman, P.W., et
al., Phytopath. Zeit. 113:165-170 (198S); Albersheim,
P.A., et al., Structure and Function_of Plant Genomes, NATO
Adv. Study Inst. Series. Plenum Publ. Corp. N.Y. pp. 293-
312 (O. Ciferri (ed.) 1982); Graham, T.L., et al., Applied
& Environmental ~icrobiology 34:424-432 (1977); ~an Loon,
L.C., Netherlands Journal of Plant Pathology 89:265-273 :
(1983); Soliman, H.N., Egyptian Journal of Phytopathology
18(1):35-45 (1986); Salt, S.D., et al., Physiological &
Molecular Plant Pathology 28(2):287-297 (1986); White,
R.F., Virology 99:410-412 (1979); Gianinazzi, S. & B.
Kassanis, Journal of General Virolo~y 23:1-9 (1974); and
van Loon, L.C., Virology 80:417-420 (1977)). These
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inducers included such materialsi as chitin,
oligosaccharides and polysaccharides, or other components
of cell walls, in addition to chemicals such as salicylic
acid.
Although those abiotic inducers were advantageous
in that they were not likely to cause disease, there were
numerous disadvantages inherent in the use of abiotic
inducers including the ability to induce resistance only
against a very limited spectrum of pathogens and the need
for multiple applications. For example, application of
oligosaccharides would induce resistance only against
organisms with cell walls that were structurally similar to
the inducing compound used. In addition, certain abiotics
(e.g. HgCl2) were potentially hazardous to work with and
the "cure~' caused damage to the plant which was worse than
the disease. Other abiotics (e.g. W light) were
impractical to apply.
Accordingly, there remains a need for biological
control of plant pathogens. Specifically, there is a need
for a method for enhancing the natural mechanisms for
resisting diseases present in commercially-valuable plants
using an organism that will not harm the environment, will
not harm the plant, is capable of living within the plant,
requires only a single application, and will enhance
protection against a broad spectrum of disease organisms.
SUMMARY OF THEI NVENTION
The present invention overcomes the problems and
disadvantages of the prior art by pro~iding of method of
enhancing disease resistance in commercially-valuable
plants, comprising providing an endophytic organism which
is capable of being harbored wi~hin the plant and which
creates no visible manifestations of disease and t in one
embodiment, crea~es no ill effects on the host plant. This
organism is introduced into the plants to enhance
protection against a wide spectrum of diseases. In
addition, the present invention provides a method of
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enhancing protection in commercially-valuable plants using
a vascular-inhabiting endophyte, i.e., one that lives in
the vascular tissues of the plant. In another em~odiment,
the invention relates to a method of enhancing protection
using a vascular-inhabiting endophyte that is a gram
positive bacterium. In still another embodiment, the
present invention relates to a method of enhancing
protection in commercially-valuable plants using an
endophytic organism that lives in the vascular-inhabiting
system of the plant, is gram positive, and is fastidious.
The invention also provides for a method of enhancing
protection in commercially-valuable plants using an
endophytic organism known as Clavibacter xYli subsp.
cynodontis ~Cxc).
Additional objects and advantages of the invention
will be set forth in part in the description which follows,
and in part will be clear from the description, or may be
learned by practice of the invention. These objects and
advantages of the invention will be realiæed and obtained
by means of the methods particularly pointed out in the
appended claims.
It is to be understood ~hat the general description
above and the following detailed description and drawings
are exemplary and explanatory only and do not limi~ the
invention as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated
in and constitute a part of this specification, illustra~e
several exemplary embodimen~s of the invention and,
together with ~he description, serve to explain the
principles of the invention.
Fig. 1 is a graph that depicts the effect of Cxc
inoculation on leaf area of tobacco (varie~y C319)
challenged fourteen days post-inoculation with tobacco
mosaic virus (TMV).
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WO90/13224 PCr/US90/02240
.
_ 5 _ ~ ~ ~3~5
FigO 2 is a graph that depicts the effect of Cxc
inoculation of leaf area of tobacco (variety C319)
challenged twenty days post-inoculation with TMV.
Fig. 3 is a graph that depicts the effect of Cxc
inoculation on leaf weight of tobac:co (variety C319)
challenged fourteen days post-inoculation with TMV.
Fig. 4 is a graph that depicts the effect of Cxc
inoculation on leaf weight of tobacco (variety C319)
challenged twenty days post-inoculation with TMV.
Fig. 5 is a graph that depicts the titer of
Pseudomonas syrinqae pv. tabaci titer in Cxc-inoculated
tobacco (~ariety Ky-14) leaves.
DESCRIPTION OF THE PREFERRED EMBODI~MENTS
Reference will not be made in detail to the
currently preferred em~odiments of the invention, examples
of which are illustrated below and in the accompanying
drawings.
As used herein, "endophyte-enhanced protection~' is
defined as the reduction of disease in plants resulting
from the introduction of an endophyte into plants. The
present invention is not limited by the manner in which the
endophyte enhances protection of the plant against disease,
nor, as discussed more fully below, by the method of its
introduction into plants.
Unlike the induced resistance previously described
by the prior art, ~he endophytes of the present invention
do not act as pathogens in the host plant. The endophytes
are organisms that are capable of being harbored within the
plant but create no visible manifestations of disease and,
in one embodiment, have no ill effects on the host plan~.
The endophytic organisms of the present invention
may also be referred to as organisms which are capable of
entering into an endosymbiotic relationship wi~h a plant
host. The endosymbiotic relationship is one in which the
organism actually exists wIthin and may spread throughout
all or a por~ion of the host plant, without causing any
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WO90/13224 PCr/US90/02240
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significant adverse effect on the host plant. The
endosymbiotic relationship of an endophyte with a host
plant in the present invention is not limited by the nature
of the relationship and may include mutualistic and
commensalistic endophytic organisms.
The endophytes used in the method of the present
invention are contained within the plant body. In a
preferred embodiment, the endophytes are contained within
the vascular system of the plant or, in an alternative
embodiment, within the intercellular spaces of the plant.
In another embodiment, the vascular-inhabiting or
intercellular-space-inhabiting endophytes are gram-
positive. "Gram-positive~' refers to a classification of
microorganisms based on the components of the cell wall as
that term is described by Davis et al. in Microbiolo~Y, 3rd
ed., (1980), specifically incorporated herein by reference. -
In still another embodiment of the invention, the
gram-positive vascular-inhibiting endophytes are fastidlous
in nature. As used herein, the term "fastidious" refers to
organisms having complicated nutritional requirements, as
that term is defined by McCoy, R.E., in ~Chronic and
insidious disease: The fastidious vascular pathogens,~
PhYtopathoqenic ProkarYotes (Mount M.S. and Lacy, G.H.,
eds. 1982), specifically incorporated herein by reference.
In still another embodiment, the present invention
relates to endophytes of ~he Coryneform family as that term
is defined by M.J. Davis in Annual Review Phytopathology
24: 115-40 (1986), specifically incorporated herein by
reference. In another embodiment the present invention
relates to the genus Clavibacter. In a particularly
preferred embodiment, the invention relates to the
endophyte known as Clavibacter xyli subsp. cynodontis
(hereinafter "Cxc''), as that term is defined by ~.J. Davis
et al. in International Journal of Systematic Bacteriology
34(2):107-117 (April 1984), specifically incorporated
herein by reference.
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WO90/13224 PCT/US90/02~0
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The present invention contemplates the introduction
of live endophytes capable of being harbored within the
plant host. The endophytes of the present invention may
multiply within the plant host but the present invention is
not limited to endophytes that multiply within the host.
The endophytes of the present invention may be
unmodified or modified or formulated with other components
to provide beneficial properties in addition to enhanced
protection. ~odification of endophytes is accomplished by
techniques that are known to those of ordinary skill in the
art. Any means of modification and any modification of
- endophyte~ are specifically contemplated by the present
invention.
The endophytes used in the method of the present
invention may be modified, for example, by mutagenesis or
recombinant techniques known to those of ordinary skill in
the microbiology and molecular biology art in light of the
teachings contained herein. The endophyte may be modified
by the induction and isolation of mutant strains effective
in protecting plants against disease. The DNA of the
endophytes may be modified by the addition of DNA that
codes for the production of particular compounds, including
but not limited to proteins, antibiotics, and other
biochemical compaunds. Thus, the endophyte could, in
addition to enhancing protection, provide agricultural
chemicals that might benefit the plant. On such method for
~he production of such endophytes is pro~ided copending in
United States Patent Application No. 166,819 (filed ~arch
3, 1988), No. 266,232 ~filed October 10, 1988), and No.
266,221 filed October 10, 1988), all of which are commonly
assigned to the assignee of the present invention and are
incorporated specifically herein by reference.
Alternati~ely, endophytes may be modified by
mutagenesis or recombinant techniques to produce inducer
compounds, such as, for example, dihydroxy benzoic acid or
beta-ionone. The techniques for these modifications are
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WO90/132~4 PCT/US90/02~0
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similarly within the routine skill of one ordinary skill in
the art in light of the teachings contained herein.
In addition, plant protection provided by the
present invention may be enhanced by formulating the
endophyte with one or more abiotic inducers. The
techniques to select abiotic inducexs and de~elop
formulations including them are within the routine skill of
those of ordinary skill in the art in light of the
teachings contained herein.
The modified, unmodified or formulated endophytes
may be introduced to the plants by any technique known to
those ordinary skill in the art. The method of endophyte
introduction does not in any way limit the present
invention. Introduction techniques, which vary with the
plant host, include, bu~ are not limited to, latex plugs,
slow releases, root drips for transplanted plants, abrasive
sprays, needle or needless injection, pressure injection
and the like.
In a preferred embodiment, the endophytes are
introduced by stem stabbing. "Stem stabbing" refers to the
introduction of endophytes by wounding the plant and ; -
delivering the endophytes to that wound. A preferred
method of stem stabbing involves a scalpel or other sharp
instrument that is first coated wi~h an endophyte and then
used to simultaneously wound and deliver the organism.
In another embodiment, the endophytes are
introduced to plants by stem injection. "Stem injection"
refers to the introduction of organisms into the stem of
the plant via a puncture created by a needle of, for
example, a tuberculin intradermal syringe. In one
preferred method, the needle of the syringe, containing the
endophytic organisms to be introduced, i~ gently pushed ~ -
into the stem and the contents of the syringe gently and
slowly injected into the stem.
In another embodiment, the endophytes are
introduced to plants either by injection into the petiole
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WO90/13224 PCT/US90/02240
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by needle or by deposition onto a previously broken
petiole.
In still another embodiment, the endophytes can be
introduced by intercellular infiltration, where a
suspension of endophytes is injected into the intercellular
spaces of a leaf.
In still another embodiment of the invention, the
endophytes may be introduced by inoculating the seeds of
the plant with the endophytes. The method of seed
inoculation is provided in co-pending United States Patent
Application No. 194,247, filed May 16, 1988, to Jed W.
Fahey, incorporated specifically herein in its entirety by
reference.
The invention relates to enhanced protection in all
commercially-valuable plants. Persons of ordinaxy skill in
the art are generally familiar with agriculturally-valuable
plants. These include the horticultural plants, such as
those producing fruits, vegetables, flowers and ornamental
trees and plants. In addition, commercially-valuable
plants include agricultural trees and plants such as field
and row plants. Field and row plants include, but are not
limited to, corn, sorghum, wheat, barley, oats, rice,
tomato, potato, cabbage, broccoli, melons, cucumbers and
related plants. In another embodiment, commercially-
valuable plants encompass plants of forestry. This list is
exemplary only and does not in any way limit the
application of the present invention.
In accordance with the present invention, the
protected plants become resistant to one or more of a broad
spectrum of diseases including, but not limited to,
mildews, rusts, smuts, rots, scabs, spots, blights, blasts,
decay, damping-off, leaf rolls, vascular wilts, warts,
galls, yellows, cankers, mosaics, ring spots and other
stunting, dwarfing or disfiguring plant diseases. These
diseases include those caused by bacteria, viruses, and
fungi and othex biotic pathogens. In a preferred
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WO90/13224 PCT/US90/02240
~ ~S3~43'3
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embodiment~ the plants are resistant to tobacco mosaic
virus, potato viruses X and Y, Pseudomonas svrin~a_ pv.
tabaci, Clavibacter michiqanense subsp. michiqanense and
Fusarium oxys orum f.sp. melonisO
The invention will be further illustrated by the
following examples, which are intended to be purely
exemplary of the invention.
EXAMPLE 1
LOCAL LESION (HYPERSENSITIVE) RESPONSE IN ENDOPHYTE~
INOCULATED TOBACCO PLANTS FOLLOWING CHALLENGE
WITH TOBACCO MOSAIC VIRUS
Plants of Nicotiana tabacum L. cv. 'Ky 14', a
variety hypersensitive to tobacco mosaic virus (hereinafter
"TMV"), were planted in one gallon pots in the greenhouse.
Thirty days after sowing, the plants were selected for
uniformity. Plants were randomly assigned as either `
control plants or treatment plants.
A. Pre~aration of the Endophyte ~Cxc !:
For six days at 28C + 3C, Cxc was grown on SC
Media, consisting of 1000 ml distilled water; 17 g cornmeal -
agar; 8 g papaic digest of soy meal; 1 g K2HPO4; 1 g
KH2P04; 0.2 g ~gS04 7H20; lS mg (15 ml of a 0.1 percent
solution in 0.05N NaOH) bovine hemin chloride; 2 g (10 ml
of a 20 percent aqueous solution) bovine serum albumin
fraction 5; 0.5 g (1.0 ml of a 50 percent aqueous solution)
glucose; and 1 g (free basef 10 ml of a 10 percent aqueous
solution) cysteine. After incubation, the cells were
washed and suspended in 10 ml sterilized tap water.
Suspensions of Cxc cells were centrifuged at 6000 rpm for
15 minutes and resuspended in sterile water or phosphate
buffered saline (PBS). Bacterial concentration was
determined spectrophotometrically at 600 nm and adjusted to
ca. 10 bacteria/ml.
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WO90/13224 P~TIUS~0/02~0
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B. Inoculation with the EndoPhvte tCxc):
The treatment plants were inoculated with solutions
containing Cxc as follows:
1. - Stem Inoculations
a) Iniection of Cxc cells into the stem 4 cm
above the soil surface by hypodermic
syringe.
b) Stabbinq of Cxc into the stem by sterile
scalpel blades containing Cxc scraped
from streaked plates where the scalpel
tip was inserted completely through the
stem.
2. - Petiole Injections
Injection of Cxc into the petiole by
hypodermic syringe.
3. - Intercellular Infiltration.
Injection of ca. 10 ul of bacterial suspension
into the intercellular space of the leaf anima
to create water soaking; eight injections per
leaf, with hypodexmic syringe.
In addition, control plants were inoculated with
control solutions, i.e., the s~me solutions as above except
that Cxc was absent.
C. Inoculation with the Challenae Or~anism:
Two weeks later, partially purified suspensions of
strain U-1 of TMV in phosphate buffered saline were used
for all challenge inoculations, as in R.W. Fulton,
~Nicotiana As Experimental Virus Hosts, "Nicotiana
Procedures for Experimental Use - Technical_Bulletin No.
1586 (U.S.D.A. P.D. Durkin, ed., 1979), specifically
incorporated herein by reference. A gauze pad was soaked
in the ~NV inoculum and rubbed onto all expanded leaves
following a light dusting with 600 mesh carborundum, an
abrasive powderl to facilitate viral infection.
As set forth in Table 1, prior inoculation of
tobacco variety RY-14 with Cxc resulted in a consistent
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WO90/t3224 PCT/US90/02240
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reduction of TMV lesion numbers. Accordingly, prior
inoculation with Cxc resulted in a dramatic reduction of
the hypersensitive response compared to controls.
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WO90/13224 PCT/U~90/02~40
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EXAMPLE 2
LOCAL LESION (HYPERSENSITIVE) RESPONSE IN
ENDOPHYTE-INOCULATED TOBACCO PLANTS FOLLOWING
C~L~LLENGE WITH TOBACCO MOSAIC VIRUS
In the same manner as Ex~mple 1, in a field
experiment, Cxc-inoculated tobacco plants t~ariety KY 14),
planted in a randomized complete ~lock design with five
replications, exhibited a reduction in lesion number over
controls when plants were challenged at fourteen days and
twenty days (two separate readings) post-inoculation (see
Table 2 below). All plants (both control and experimental)
challenged at thirty-one days showed such low numbers of
lesions that comparisons between treatments is not valid,
likely due to environmental conditions in the field. :~
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Table 2
Effect of Cxc Inoculation on TMV Lesion Number
in Field-Planted Tobacco Variety KY 14
. . . ~
Mean ~ of lesions/100 sq cm leaf
Method of
Inoculation Days After Cxc Inoculation
14 20 20 31
ExperimentalStem Stab 60 49 20 8.4
with Cxc
Stem Inject 60 41 14 6.9
with Cxc
_______________________________________________ ______ ___
ControlS~em Stab 60 57 24 8.1
with Water
Stem Inject 63 52 23 8.7
with Water
Unioculated 85 58 29 6.7
Control
LSD = 32.4 13.6 9.85 4.5
N = 100 68 80 100
*L$D refers to Least Significant Difference
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W090/13224 PCr/US90/02~40
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EXAMPLE 3
SYSTEMATIC RESPONSE IN ENDOPHYTE-INOCULATED
TOBACCO PhANTS FOLLOWING CHALLENGE WITH TMV
Using the protocol of Example 1, sixty plants of
tobacco variety Coker 319 (C319) were planted in the
greenhouse. Af~er five weeks of growth, uniform plants
were selected and subjected to the following treatments:
1) untreated control
2) stem stab with water (control)
3) stem stab with Cxc
4) stem injection with Cxc.
The entire experiment was repeated three separate
times.
Plan~s were inoculate~ with Cxc at the stage of
growth when two true leaves had formed. Fourteen days
later, the plants were challenged with TMV by inoculation
of true leaves four and five (counted from the soil line).
Twenty days later, leaves numbered 5, 6, 7, and 8, counted
from the challenged leaf, were removed and leaf area and
leaf fresh weight were assessed. Leaf fresh weight was
determined by removing any adherent water or debris and
weighing the entire leaf and subtending petiole. Nine days
later, plants were re-assessed by measuring leaf area and
fresh weight for leaves numbered 9, 10, 11, and 12. In
addition, plant height was determined by measuring total
height of the plant from the 50il line to the uppermost
leaves and plant weight was scored as the weight of the
above-ground portion of the plant.
In two of the three replications, Cxc-inoculated
plants exhibited greater leaf area and leaf weight than
their respective controls. Table 3 sets forth results from
one of those replications in which the leaf areas of leaves
numbered 7, 8, 9, 10, 11, and 12, from Cxc inoculated
plants, were significantly greater than their respective
controls.
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WO90/13224 PCT/VS90/02240
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Leaf fresh weight exhibited a similar pattern, as
depicted in Table 4. Leaves 7, 8, 9, 10, 11 and 12 from
Cxc-inoculated plants exhibited significantly greater fresh
lea weight than control plants.
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WO90/13224 PCT/US90/02240
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Similarly, as depicted in Table 5, inoculated
plants exhibited increased plant height and weight compared
to controls.
Table 5
Effect of Cxc Inoculation on Plant Height and Weight
of Tobacco Variety C-319 Challenged with TMV (REP#1)
Method of Plant HeightPlant Weight
Inoculation (cm) (g)
Experimental Stem Stab 46 211
with Cxc
S~em Inject 43 189
with Cxc
____________________________________ .______________ _______
Control 5tem Stab 38 161
with Water
Uninoculated 42 166
Control
.
LSD = 5 . 5 25
EXAMPLE 4
SYSTEMIC RESPONSE IN ENDOPHYTE- INOGUI~ATED :
TOBACCO PLANTS FO~LOWING CHALLENGE WITH TMV
In the same manner as Example 3, in three field -
experiments, Cxc-inoculated plants exhibited significantly
greater leaf area and leaf weight when challenged fourteen
and twenty days post inoculation. As shown in Figures 1
and 2, the a~erage leaf area of Cxc-inoculated plants was
greater than control plants. This pattern was exhibited
when challenge occurred at either fourteen days (Figure 1)
or twenty days (Pigure 2) after inoculation. Similarly,
leaf weight of Cxc-inoculated plants challenged fourteen
(Figure 3) or twenty days post inoculation (Figure 4) was
significantly greater than that of control plants.
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EXAMPLE 5
SYSTEMIC RESPONSE IN ENDOPHYTE-INOCULATED
TOMATO PLANTS FOLLOWING CHALLENGE WITH TMV
Using the protocol of Example 1, tomato plants
(Lyco~ersicon esculentum cv. Marglobe), susceptible to TMV,
were planted in a greenhouse, tran.splanted into one gallon
pots approximately six days later, and allowed to continue
to grow in the greenhouse. This experiment was replicated
twice. In both replications, plants were inoculated with
Cxc one week after transplanting. All plants were
challenged with TMV approximately three weeks after Cxc
inoculation.
Approximately 25 days after challenge, the plants
were assessed for the number of flowers produced, the
number of fr-liting bodies produced, and plant height.
Table 6 sets forth the effect of Cxc inoculation on
flowering and fruiting. Flowering scores were higher for
inoculated plants than controls. Similarly, inoculated
plants exhibited greater fruiting than did controls.
Table 6
Effect of Cxc on the Flowering and
Fruiting of TMV Challenged Tomato
~ .
FLOWERING FRUITING
TREATMENT SCORE SCORE
... ._ _
Experimental Stem stab 2.3a 1.4a
with Cxc
Stem inject 1.9 1.2
with Cxc
___________________________________________________________
Control Stem stab 1.5 1.0
with Water
Stem in~ect 1.8 1.0
with Water
a = Scores:
3=100% of plants flowering or fruiting
2=50 % of plants flowering or fruiting
1=0 ~ of plants flowering or fruiting
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When TMV challenge of tomato plants was performed
in the field, Cxc-inoculated plants exhibited an increase
in yield over control plants, as set forth in Table 7.
Table 7
Effect of Cxc Inoculation on Yield
of Tomato Plants Following Challenge with TMV
Method of Tomato Yield
Inoculation (Kg)
. . .
Experimental Stem Stab 2.8
with Cxc
Stem Inject 3.3
with Cxc
_________________________ _____________________________ __
Control Stem Stab 2.3
with Water
Stem Inject 2.7
with Water
Unioculated 2.7
Control --:
LSD= .6
Similarly, when the quality of tomatoes at harvest
time was assessed, as set forth in Table 8, fruit from ~he
Cxc-inoculated plants was of superior quality. In
contrast, ratings for controls were predominantly in th~
lowest quality categories (three and four).
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Table 8
Effect of Cxc Inoculation on Tomato Fruit Quality
Following TMV Challenge with TMV- -
Quality Categorya
Method of ----~
Inoculation lb 2 3 4 Average
Experimental Stem Stab 0% 12~i 80% 8~ 3.0
with Cxc
Stem Inject 0% 27% 68% 5% 2.7
with Cxc
________________________~__________________________________
Control Stem Stab 0% 0% 62% 38% 3.4 .:
with Water
Stem Inject 0% O~i 59% 41~i 3.4
with Water
Uninoculated 0% 4% 48% 48% 3.4
Control
.
a = % of total plants in each quality cate~ory
b = Quality categories:
1=mostly red :
2=half red and half green : :
3=mostly green and large
4=mostly green but small ~ .
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EXAMPLE 6
SYSTEMIC RESPONSE IN ENDOPHYTE-INOCULATED POTATO
PLANTS FOLLOWING CHALLENGE WITH POTATO VIRUSES
In a field experiment, one hundred and eight potato
plants (Solanum tuberosum cv. Kennebec) were planted by
hand in a randomized complete block design with 3
replications.
After appxoximately seventeen days of growth,
plants were treated with Cxc, introduced by either stem
injection or stem stabbing, as described in Example 1
above. Con~rol plants were stem injected with water, stem
stabbed with water, or left un~reated.
Twenty days after inoculation with Cxc, the potato
plants were challenged with potato virus X (PVX) using the
same procedure used for challenging tobacco plants with TMV
as described in Example 1.
Plants were scored for flowering and for disease
severity. As set forth in Table 9, potato plants
inoculated by either injection or stabbing with Cxc
exhibited a significantly higher percentage of flowering
than did any of the three sets of control p-lants.
Similarly, inoculated plants exhibited reduced disease
severity (as evidenced by the number of discolored and
wilted leaves) than controls. Disease severity in each
plant was rated on a scale of 1-4 based on quality of
leaves, such that:
1 - less than 10% of plants' leaves exhibited
disease symptoms
2 = greater than 10% to less than 30%
of the leaves exhibited disease symptoms
3 = greater than 30~ but less than 60% of the
leaves exhibited disease symptoms
4 = greater than 60% of the leaves exhibited
disease symptoms
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Table 9
Effect of Cxc Inoculation on Potato ~lowering and
Disease Severity Following Challenge with PVX
.. _ . . ..
DISEASE RATINGa FLOWERIIIG~
___________________.____________.____________
AVERAGE
METHOD OF DISEASE
INOCULATION 1 2 3 4 RATING
Experi-Stem stab 4% 87% 9% 0% 1.9 52%
mentalwith Cxc
Stem inject 5% 76% 19% 0% 2.2 71%
with Cxc
____________ _______ ______________________________________
ControlSte~ stab 0% 64% 32% 4% 2.3 20% . .
with Water
Stem inject 0% 52% 40% 8% 2.6 24%
with Water
Uninoculated 0% 77~ 14~ g% 2.3 9%
Control
,
a = % of total plants in each disease rating
b = % of total plants flowering in each category
Example 7.
SYSTEMIC RESPONSE IN ENDOPXYTE-INOCULATED
POTATO PLANTS FOLLONING CH~LLEN&E WITH POTATO VIRUSES .
In the same manner as Example 6, 180 potato plants
(S. tuberosum. cv. Kennebec) were planted by hand in the
field. Approximately two weeks later, the plants were
randomly assigned to fi~e groups and subjected to ~ive
treatments. As above, the five treatments were: stem stab
with Cxc, stem inject with Cxc, uninoculated c~ntrol, stem ~ :
stab wi~h water and s~em inject with water. Twenty d ys
after inoculation with Cxc, all plants were challenged with
potato ~irus Y (PVY)
Thirty days later, the plants were scored for
flowering and for di ease severity. As set forth in Table
10, although there was no difference in flowering between ~ ;,
Cxc-inoculated and oontrol plants, Cxc-inoculated plants
did exhibit.a reduction in disease severity over control
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plants. Specifically, as set forth in Table 10, Cxc-
inoculated plants predominantly ranked in the lowest damage
category.
Table 10
Effect of Cxc Inoculation on Potat:o Flowering and Disease
Severity Following Challenge with PVY
~ . .
DISEASE RATINGa FLOWERINGD
___________.________________________ _________
METHOD OF AVERAGE
INOCULATION 1 2 3 4 DISEASE
RATING
-
Experi-Stem stab 22% 78% 0% 0% 1.8 65%
mentalwith Cxc
Stem inject 5% 73% 23% 0% 2.2 55%
with Cxc
_________________________________________._________________
Control Stem stab 0% 80% 20% 0% 2.2 48%
with Water
Stem inject 0% 50~ 45% 5~ 2.6 65%
with Water
Uninoculated 0% 67% 29% 4~ 2.4 46%
Control ~-
r
a = % of total plants in each disease rating
b = ~ of total plants flowering in each category
EXAMPLE 8
SYSTEMIC RESPONSE IN ENDOPHYTE-INOCULATED POTATO
PLANTS FOLLOWING CHALLENGE WITH POTATO VIRUSES
In the same manner as Example 6, 180 potato plarts
were planted in the ield and allowed to grow for
approximately two weeks. The plants were then inoculated
with Cxc, using the same five treatments as set forth
abo~e. Eighteen days after inoculation with Cxc, the
plan~s were challenged with a mixture of PVY and PVX.
Seventeen days later the plants were evaluated. As
set forth in Table 11, the Cxc inoculated plants exhibited
a greater percentage of flowering than did the control
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plants. Similarly, Table 11 depicts that the Cxc-
inoculated plants exhibited less damage due ~o viral
infection than did the control plants.
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Table 11
Effect of Endophyte-Inoculation on Potato Flowering and
Disease Severit~ Following Challenge with PVX + PVY
DISEASE RATINGa FLO~ERINGD
_________ .________________________ __________
METHOD OF AVERAGE
INOCULATION 1 2 3 4 DISEASE
- RATING
Experi-Stem stab 77% 23% 0% 0% 1.2 88
mentalwith Cxc
Stem inject 77% 23% 0% 0% 1.2 . 95%
with Cxc
___________________________________ _ _____________________ . .
Control Stem stab 4% 84%12% 0% 2.1 68%
with Water
Stem inject 12% 72~16% 0% 2.0 80%
with Water
Uninoculated 7% 86%7% 0% 2.0 42%
Control
-
a = % of total plants in each disease rating
b = % of total plants flowering in each category
EXAMPLE 9
SYSTEMIC RESPONSE OF ENDOPHYTE-INOCULA~ED TOMATO CHALLENGED
WI'rH CLAVIBA~CTER MICHIGANENSE SUBSP. MICHIGANENSE
Using the protocol of Example, tomato plants
(Lvco~ersicon esculentum cv. Marglobe) were planted in a
greenhouse and allowed to grow for twelve days.
Thereafter, plants were divided into five groups as
~ollows:
1) untreated control
2) stem stab with water
3) stem inject with water
4) stem stab with Cxc
5) stem inject with Cxc.
Eleven days later, ~he tomato plantæ were
challenged by introduction of the bacteria Clavibacter
michi~anense subsp. michiganense ("Cmm") (syn.
C rvnebacterium mLchiqanense subsp. michiqanese) grown on
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nutrient broth yeast extract agar (NBY) and incubated at
26C for approximately four days. Cell suspensions of Cmm
in distilled water were used for challenge inoculation.
Challenge inoculation was performed by breaking the petiole
and applying a suspension containing approximately 108
cells/ml to the broken area.
Eighteen days after challenge, the plants were
scored for disease severity. The severity of disease in
each plant was rated on a scale of 0 to 4 as follows:
0 = no evidence of disease
1 = 0-10% of plant wilted
a = 10%-40% of plant wilted
3 = 40-75% of plant wilted
4 = greater than 60% of plant wilted
As set forth in Table 12, the inoculated plants
exhi~ited a dramatic reduction in disease compared to
control plants.
Table 12
Effect of Endophyte-Inoculation on Tomato Disease Severity
Following Challenge with C. michi~anense subsp.
michiq~ense
DISEASE RATING , -
______________________________________________ , . .
METHOD OF
INOCULATION la 2 3 4 AVG.
_ _ .
Experimental Stem stab 33% 56% 11% 0% 1.8
with Cxc
Stem inject 22% 56% 22% 0% 1.9
with Cxc
___________________________________________________________
Control Stem stab 0% 20% 60% 20~ 3.0
with Water
Stem inject 0% 20% 80% 0~ 2.8
wi~h Water
Uninoculated 0% 0% 100%0~ 3.0
Control
.._ _ _ . . _ . .. .
a - ~ of total plants in each disease categcry
_ . _ _ . .
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EXAMPLE l0
SYSTEMIC RESPONSE IN ENDOPHYTE-INOCULATED TOBACCO PLANTS
FOLLOWING CHALLENGE WITH PSEU _MONAS SYRINGAE
pv. TABACI
Using the protocol of Example 1, tobacco plants
were planted in the greenhouse. Af~er an initial growth
period of about 1 month, in each of three randomly assigned
replications, ten plants were stem injec~ed with Cxc, ten
were stem stabbed with Cxc, five were stem stabbed with
sterilized tap water, and five were untreated.
Fourteen days after inoculation with Cxc, the
plants were challenged with Pseudomonas s~rinqae pv. tabaci
(~'Ps tabaci ). At 1, 2, 3 and 4 days after inoculation
with this bacterium, a disc was punched from the leaf
tissue between lesions. The discs were homogenized and
plated onto Kings B Agar to calculate the number of Ps.
tabaci cells per gram of leaf tissue. These numbers
provided an index of bacterial infestion.
As set forth in Figure 5, inoculated plants
exhibited a reduction in the number of bacterial cells per
gram of leaf tissue each day after inoculation.
Accordingly, it appeared that the Cxc-inoculated plants
permitted less multiplication of Ps. tabaci in leaf tissue.
This reduction of pathogen/titer in the plants has a direct
impact on the rate of spread of ~he resultant disease
(wildfire disease) in the field.
EXAMPLE 11
SYSTEMIC RESPONSE IN ENDOPHYTE-INOC~LATED MUSRMELON
CHALLENGE WITH FUSARIUM OXYSPQRUM f.sp. MELONIS
Cucumis melo Muskmelon (variety Honey Rock) plants
were grown in a greenhouse. At approximately one week
pos~-emergence, plants were subjected to one of two
treatments: hypodermal inoculation with ~ashed Cxc cells
resuspended in phosphate buffered saline ("PBS") at
approximately 108 CFU/ml; or a control inoculation using
PBS alone.- After approximately 26 days of growth, all
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plants were challenged with Fusari~ oxysPorum f.sp.
melonis by transplanting into pots containing Fusarium
infested soil. All plants were allowed to grow with a
photoperiod of about 14:10 (L:D), at 95~ relative humidity
and 23C until symptoms appeared. Two replications were
conducted.
Plants were assessed for disease se~erity and plant
dry weight. Plant weight was determined by weighing the
harvested, above-ground portions of the plant. The
severity of each disease was assessed using standard
phytopathological methods and plants were rated on a scale
of 0-5:
0 = no evidence of disease
1 = 0 - 10% of plant wilted
2 = 10% - 30% of plant wilted
3 = 30~ - 60% of plant wilted
4 = greater than 60% of plant wilted
5 = plant death.
As set forth in Table 13, Cxc-inoculated plants exhibited
greater dry weight and reduced disease severity compared to
controls.
Table 13
Effect of Cxc Inoculation on Vascular Wilt of Muskmelon
Following Challenge with Fusarium oxYsporum f.sp. melonis.
_
Mean Disease Mean
Treatment Severity Score Dry wt. (g)
_____________________ _. ___~_______________________________ .
Replication 1
Cxc-inoculated 1.7 1 85
PBS-inoculated control 3.7 1 58
Replication 2
Cxc-inoculated 2.58 4 01
PBS-inoculated control 3.75 2 15
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Other embodiments of the invention will be apparent
to those skilled in the axt from a consideration of the
specification and practice of the inventicn disclosed
herein. It is intended that the specification and the
examples be considered as exemplary only, with the true
scope of the spirit of the invention being indicated by the
following claims and their equivalents.
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