Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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BACXGROUND OF THP INVENTION
The invention described herein was made in the
course of work under a grant or award from the
Department of Health, Education and Welfare.
This invention relates to the protection of
frost-sensitive plants against frost injury and, more
particularly, to the inhibition oE ice formation in
plant tissues at moderate supercooling temperatures.
Damage to crops by frost is one of the leading
causes of loss in agricultural output due to natural
phenomenon variability in the world, to be exceeded
only by drought and flooding, pests and diseaseO It
is estimated that from 5-15% of the gross world
agricultural product may be so 105t to frost damage in
one year. In some regional areas ( i.e. counties,
valleys) the loss may approach 100%.
The greatest amount of frost damage to sensitive
crops does not occur in northern or cold climates.
Instead, it occurs at mid- and low-latitudes and at
high altitude equatorial locations where high value
food corps such as soybean, corn, orchard fruits, and
vegetables are grown. For instance, the orchards of
California, vineyards o Italy, the corn and soybeans
of Iowa, and potatoes of Ecuador all suffer damage
each year from the same phenomenon--light night frost
at temperatures from - 1C to -4C.
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It has been estimated by the United States
Department of Agriculture that about 1.5 billion
dollars of agricultural products is lost to frost
damage in the United States each year. The world-wide
total is probably in excess of 10 billion dollars.
For the most part, present frost protection
methods are centered around the principIe of
maintaining heat in a crop to keep it from cooling
below the freezing point where frost is imminent.
This is done by a variety of methods such as burning
oil or natural gas, stirring the air over crops,
sprinkling the crops with water, and covering them.
With the cost of petroleum becoming more expensive and
pressures against polluting the air with anthropogenic
wires, heating large areas of agricultural land to
prevent frost damage may become increasingly unpopular
in the future Also, these measures all require a
considerable amount of equipment, trained and
available manpower, and are capital intensive.
In addition to these physical methods, chemical
methods of frost protection for growing plants have
been attempted by application of various chemical
compounds onto the plants with the view of lowering
the temperature at which the plant tissues would
freeze. These previously proposed chemical methods
have tended to be unreliable, expensive, and
ecologically unsound.
Frost damage to plants occurs when intracellular
liquid in the plant freezes with resulting rupture of
adjacent cell walls and cell membranes. It is known
that plant tissues may supercool to temperatures of
around -6C in the absence of external ice nuclei.
The internal plant tissues do not generally initiate
ice at temperatures warmer than this -6C threshhold.
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It has recently been established that there are a
very few bacterial species which can act as
ice-forming nuclei at relatively warm temperatures,
i.e., -1C to -3C. The bacteria Erwinia herbicola
and Pseudomonous syringae have been identified as
being representative, if not the sole species, of
these bacteria acting as ice nucleants on plant
tissues.
To protect plants from fxost damage, it is
therefore desirable to have available means for
reducing the populations or otherwise inhibiting the
ice-nucleating activity of the ice-nucleating bacteria
on plant leaves, so -as to thereby recluce the
temperature at which frost injury occurs to
temperatures approaching -6C. The use of various
chemical bactericides or this purpose has not thus
far proven to be a satisfactory approach, since
besides being expensive and ecologically unsound, such
bactericides have not been species-specific to the
ice-nucleating bacteria, but instead have been
deleterious to the plants by also killing the
beneficial bacteria.
Another recently proposed approach to this
problem, as described in the Arny et al ~.S. Patent
Nos. 4,045,910 and 4,161,084,
is to apply to the plants competitive
non-ice-nucleating bacteria in an amount sufficient to
increase the proportion of non-ice-nucleating bacteria
to ice-nucleating bacteria from that normally present
on the plants, .hereby reducing the probability that
sufficient numbers of ice-nucleating bacteria will be
able to grow on the plant leaves. This approach
requires application of the competitive bacteria at a
rather substantial time prior to the onset of freezing
temperature and/or at a rather early stage of plant
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growth so as to enable the competitive bacteria to
adequately establish themselves on the plant leaves in
order to be effective. Moreover, this approach has
not been found to be fully reliable or confidently
repeatable in field trials, presumably due to an
ability on the part of the ice nucleating bacteria to
re-establish their original proportion to the
non-ice-nucleating bacteria on the plants.
SUr~ARY OF THE INVENTION
It is, accordingly, a primary object of the
present invention to provide an improved method for
protecting plants against fxost injury by inhibiting
ice formation in the plant tissues at moderate
supercooling temperatures.
Another object of the invention is to provide an
improved method for protecting plants against frost
injury in accordance with the preceding object, which
is more reliable, convenient and economical than the
prior art frost protection procedures.
A further object of the invention is to provide
an improved method for protecting plant against frost
injury in accordance with the preceding objects, which
is ecologically sound and leaves no harmful residue
which collects in the environment, and which is
harmless to plants and animals.
Still another object of the invention is to
provide an improved method for protecting plants
against frost injury in accordance with the preceding
objects, which can suitably be emploved at any stage
of plant growth and either relatively shortlv prior to
the onset of freezing temperature or as a long-term
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prophylactic treatment at the beginning of a growing
season.
A still further object of the invention is to
provide an ice nucleation-inhibiting composition for
topical application to plants which specifically
inhibits the ice nucleating activity of ice-nucleating
bacteria normally present on plants without harming
any other living organism, and which is ecologically
sound and leaves no harmful residue which collects in
the environment.
Yet another object of the invention is to provide
an ice nucleation-inhibiting composition in accordance
with the preceding object, which is suitable for being
conveniently and economically sprayed onto plants by
means of conventional irrigation sprinklers or
insecticide foggers.
The above and other objects are achieved in
accordance with the present invention by means of
non-phytotoxic virulent bacteriophages which are
species~specific to the ice-nucleating bacteria
normally present on plants. When topically applied to
frost~sensitive plants, at a time sufficientlv prior
to the onset of freezing temperature and in a
sufficient concentration, such bacteriophages protect
the plants against frost injury by inhibiting the
ice nucleating activity of the ice-nucleating
bacteria, thereby reducing the temperature at which
frost injury occurs to temperatures approaching -6C.
Due to their species specificity, the bacteriophages
selectively attach only the ice-nucleating bacteria,
and are harmless to any other living organism. They
are derived from the natural ecosystem to which they
are being applied, and hence their application to
plants is ecologically sound and leaves no harmful
residue which collects in the environment.
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The bacteriophages in accordance with the present
invention, along with a suitable non-phyto~oxic
carrier, may conveniently and economically be sprayed
onto the plants by means of conventional irrigation
sprinklers or insecticide foggers. Application may
suitably be carried out at any stage of plant growth,
as late as 24 hours prior to the onset of freezing
temperature, or at the beginning of a growing season
as a long-term prophylactic treatment. After initial
application, the population of the bacteriophages will
grow to the limits of its host population or until
other natural factors limit such growth.
DESCRIPTION OF PREFERRER F~MBODI~IENTS
The ice nucleation-inhibiting bacteriophages in
accordance with the present invention are derivable
from various local plant material sources, such as
grass clippings or other leaf debris, and may be
isolated from these sources by viral enrichment
procedures employing isolates of any of the various
host species of ice-nucleating bacteria, e.q., Erwinia
herbicola or Pseudomonus syrin~ae. Such bacterial
isolates are readily obtainable from various culture
collections throughout the country, or may be derived
rom various plants by the well known dilution plating
technique.
In the general procedure for isolating the ice
nucleation-inhibiting bacteriophages for use in the
present invention, the plant material used as the
phage source is incubated with a high concentration
( e.g., about 109 101 cells per gram of plant
material) of the host ice-nucleating bacterial
isolate, so that the bacteriophages specific to the
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host species will have enhanced and preferential
growth. After a suitable incubation period, e.g.,
overnight, the incubation mixture is clarified by
centrifugation, and chloroform is then added to the
supernatant broth solution so as to kill all the
bacterial species therein. Samples of the resulting
solution, containing a mixture of bacteriophages, are
then plated on a high concentration ( erg., about
108-109/ml) of the host ice-nucleating bacterial
isolate, using the standard agar overlay method,
resulting in plaques being formed by the
bacteriophages of interest. These plaques are then
picked with sterile toothpicks, put in a sterile broth
and again replated, and single plaques picked.
The thus isolated bacteriophage is then purified
from the plaque by standard differential
centrifugation procedures. The bacteriophage is first
extracted from the plaque with a suitable buffer,
e.q., 0.002 M phosphate buffer, pH 7.0, containing
0.001 I MgSO4 and saturated with chloroform to kill
all bacterial species. After removing bacterial
debris from the extract, eOg., by centrifugation at
3,000 x g for 10 minutes, the bacteriophage is
sedimented at higher centrifugation conditions, e.g.,
15,000 x g for one hour.
The resulting isolated and purified bacteriophage
is suitably stored in sealed ampules at 4C in a
suitable buffer, e.q., 10 3 M phosphate buffer, pH
-
7.0, containing chloroform or 0.1 percent sodium azide
to prevent bacterial contamination.
The general procedure described above has been
used to isolate a number of virulent bacteriophage
strains which are representative of ice
nucleation-inhibiting bacteriophages suitable for use
in the present invention. These strains have been
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designated Erh 1, Erh 2, Erh 3, Erh 4, Erh S, Erh 6,
and Erh 7 (species-specific for Erwinia herbicola),
and Pss 1, Pss 2, Pss 3, Pss 4, Pss 5, Pss 6, Pss 7,
Pss 8, Pss 9, and Pss 10 (species-specific for
Pseudomonas ~yringae). Bacteriophages Erh 1, Pss 8,
and Pss lO are preferred, and bacteriophage Erh 1 is
particularly preferred, for use in this invention.
The relative efficacy of any given bacteriophage
strain, isolated and purified in accordance with the
procedure described above, in inhibiting the
ice-nucleating activity of its host species of
ice-nucleating bacteria, can be readily measured by
means of the freezing drop method described by Vali
tmos. Sci., Volume 28, pages 402-~09, 1971).
rrhis testing procedure is carried out by drawing
portions of the sample for testing into a sterile
plastic syringe capped with a sterile needle and using
the syringe and needle combination to make equal-sized
drops on a thermally controlled cold stage. The drops
are positioned on a thin square of mylar or aluminum
foil held on a cold surface with a light coating of
mineral oil. Prior to the application of drops, the
foil is coated with silicone resin using paper tissue,
to assure that ice nucleation events are not
influenced by extraneous nuclei on the foil surface.
The silicone also causes drops to "bead up" forming
hemispheres. Twenty to lO0 drops of 0.01 cm are
used for each test. The temperature of the sample is
then gradually supercooled, and the freezing of the
drops is detected visually based on changes of the
drops from clear to opaque upon freezing. From the
observed freezing temperatures of the drops, ice
nucleous activity spectra can be constructed. By
comparing the freezing spectrum of a culture of the
host species of ice-nucleating bacteria treated with
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the bacteriophage, with the freezing spectrum of the
untreated culture, a good measure of the ice
nucleation-inhibiting efficacy of the bacteriophage is
obtained.
y following the general isolation, purification,
and efficacy testing procedures described herein, and
varying the plant material source and/or the host
ice-nucleating bacteria species or isolate, it will be
readily apparent that an infinite numoer of
non-phytotoxic virulent bacteriophage strains can
readily be obtained; and their relative efficacies in
the practice of the present invention readily
ascertained. It will be understood that any and all
of such bacteriophage strains having the ability to
i.nhibit the ice-nucleating activity of ice-nucleating
bacteria, are contemplated as being within the scope
of the resent invention, and that any specific
description herein relative to the exemplified
bacteriophages is given for illustrative purposes
only, and is not be to considered in any way limiting.
For use in the practice of the present invention,
the ice nucleation-inhibiting bacteriophages,
preferably in a senescent state, are most
advantageously employed in admixture with a
non-phytotxic carrier therefor. Particularly suitable
ice nucleation-inhibiting compositions in accordance
with the present invention, are suspensions of the
bacteriophages in an aqueous medium, preferably
buffered, e.g., with phosphate salts, to a pH within
the range of prom about 6.5 to about 7.5. Such
aqueous medium may suitably contain one or more
additives, such as nutrients or protective agents for
the bacteriophages. The incorporation into the
aqueous medium of gelatin, in a concentration of about
0.1 percent by weight, has been found to be most
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advantageous in protecting the bacteriophages against
surface tension denaturation~
The concentration of bacteriophage in the ice
nucleation~inhibiting compositions in accordance with
the present invention, will generally be at least
about 10 phage particles per ml. Preferably, such
bacteriophage concentration will be within the range
of fxom about 5 x 101 to about 2 x 1013 phage
particles per ml, which represents a concentration of
the bacteriophage by a factor of up to about 108
greater than normally found in nature. The optimal
concentration of bacteriophage in the composition will
be based upon the population of ice-nucleating
bacteria present on the plants to be protectedj and
could be readily determined by bacterial count tests.
The ice nucleation~inhibiting compositions of the
present invention may suitably be topically applied to
the plants by spraying, fox example, by means of
conventional irrigation sprinklers, insecticide
foggers, or small hand sprayers. The composition
should be sprayed on the plants in an amount
sufficient to wet the plant leaves, typically in an
amount of up to about 0.1 ml per cm2 of leaf
surface. For a typical leaf, assuming good wetting,
and a concentration of ice-nucleating bacteria of
106 cells per cm2 of leaf surface, for optimum
frost protection, the application should be in an
amount of at least about 101 phage particles per
cm of leaf surface. This figure would have to be
adjusted for higher or lower bacterial counts.
Concentrations greater than indicated by bacterial
counts can be used, but other than providing a safety
factor, will generally offer no significant gains in
protection.
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Satisfactory frost protection can be obtained
with the ice nucleation-inhibiting compositions of the
present invention, regardless of the stage of plant
growth at the time of the application. Given ideal
conditions, frost protection could take place in the
space of only a fell hours following application. More
practically, however, application shou3d take place at
least 24 hours prior to the onset of freezing
temperature. After initial application, the
population of the bacteriophages will grow to the
limits of its host population or until other natural
factors limit such growth. The fact that the
bacteriophages will propogate on the plants enables
the application to be made at the beginning of a
growing season as a long-term prophylactic treatment.
he ~rost-sensitive plants protectable against
frost injury by means of the ice nucleation-inhibiting
compositions of the present invention, include a wide
variety of high value food crops and ornamental
plants, such as, for example, beans, corn, tomatoes,
pumpkins, potatoes, soybeans, a pull range of citrus
fruits, apples, pears, hard nuts, and a full range of
cereal crops. Moreover, since the bacteriophage
inhibitors of the present invention are harmless to
any living organism other than the specific
ice-nucleating bacteria on which they are predators,
and since these bacteriophages are part of the natural
eco-system to which they are being appled, it is
ecologically sound to apply them to any and all crops
destined for human consumption. Furthermore, these
bacteriophages leave no residue that collects in the
environment like many pesticides do, nor do thev have
any known side effects to plants or animals.
While not intending to be bound by or limited to
any particular theory of the mechanism of action of
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the ice nucleation inhibitors of the present
invention, it is believed that the bacteriophage
selectively attacks and kills its host species of
ice-nucleating bacterial by first attaching by its
tail to the outside of the cell wall of the bacterium,
and then releasing its DNA or RNA gene component from
its head down its hollow tail tube and through the
bacterail cell wall into the interior of the bacterial
cell. The gene component then replicates the original
bacteriophage inside the bacterial cell. During such
replication, the ice-nucleation sites present on the
bacterial cell wall become deactivated or blocked from
inside the cell wall. Continued replication of phage
particles within the cell Bills the bacterium by
causing it to burst, thereby releasing a many fold
increased number of bacteriophages. it this point the
bacteriophages go on to infect more bacterial in ever
increasing numbers, until they have infected all the
ice-nucleating bacteria available to them. In so
affecting the ice-nucleating bacteria, they reduce
their potential for acting as ice nuclei.
The ice nucleation-inhibiting compositions of the
present invention may suitably be applied to plants in
conjunction with other known frost prevention
compositions to maximize the benefits and advantages
of each technique. For example, the competitive
non-ice-nucleating bacteria technique described in the
Arny, et al., U.S. patent numbers 4,045,910 and
4,161,084, incorporated herein by reference, has not
generally been found to be fully reliable or
confidently repeatable in field trials, presumably due
to an ability on the part of the ice-nucleating
bacteria to re-establish their original proportion to
the non-ice-nucleating bacteria on the plants. The
species specificity of the ice nucleation-inhibiting
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bacteriophages of the present invention to
ice-nucleating bacteria, would enable them to
compatibly be used in conjunction with the Arny, et
al., technique, such as, for examp:Le, by including
non-ice-nucleating bacteria suspended together with
the bacteriophages in the same composition in an
amount sufficient, when applied to the plants, to
increase the proportion of non-ice-nucleating bacteria
to ice-nucleating bacteria from that normally present
on the plants. The combined effect of such
composition would be to kill off the ice-nucleating
bacteria on the plants and simultaneousIy replace them
with non-ice-nucleating bacteria, thereby
substantially decreasing the probability of the
ice-nucleating bacteria re-establishing their original
proportion to the non-ice-nucleating bacteria on the
plants.
The invention is further illustrated by way of
the following examples.
_xample
mploying fresh grass clippings as the source
material, and Erwinia herhicola subspecies ananas
~ATCC 8366) maintained on 1.2 agar slants containing 5
grams tryptone, 2 grams yeast extract and 25 grams
glycerol per litre of water, as the host species of
ice-nuc~eating bacteria, bacteriophage Erh 1 was
isolated and purified by means of the following
procedure.
Twenty-five ml of fresh broth culture of the host
bacterial species, at a concentration of about 2 x
10 /ml was added to 1-2 grams of the fresh grass
clippings. The inoculated cultures were shaken over
night at 23C. and clarified by centrifugation at
2,000 x g. Chloroform was added to the supernatant
broth solution, which as stored in the cold. Various
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samples of this solution were then plated on the host
bacterial species using the standard agar overlay
method on the tryptone-yeast extract-glycerol medium
using 1.2 percent agar in the base layer and 0.5
percent agar in the top layer. 0.1 ml of one-day-old
room temperature culture of the host bacterial
species, washed off fresh slants and diluted to 2 x
108/ml, was used Jo innoculate the soft outer layer.
Eight different types of plaques were picked with
sterile toothpicks, put in a sterile broth and again
replated, and single plaques picked. A variety of
phage plaque types were observed, and electron
micrographs of the particles were obtained. The most
common type of plaque was small and clear, but turbid
plaques were also observed. Many of the phage
particles looked like Tl or from the E. coli system
with long thin tails and with heads which typically
were 7 8 nm wide. One particular phaqe isolated had a
very different type of morphology with an elongated
rod-like head and a short complex tail with a base
plate. This phage (Bacteriophage Erh 1) was selected
for further purification. The bacteriophage was
extracted from the plaque with 0.002 M phosphate
buffer, pH 7.0, containing 0,001 M MgSO4 and
saturated with chloroform. Bacterial debris was
removed by centrifugation at 3,000 g for 10 minutes,
and the bacteriophage was readily sedimented in one
hour at 15,000 x g in the angle centrifuge.
Concentrated suspensions of the phage could be readily
filtered through 0.6 Nucleopore filters (95~ yield)
but passed poorly (8% yield) through 0.47 Millipore
filters. The phage particles were stored in 10 3 M
phosphate buffer, pH 7.0, and were unaffected by the
addition of chloroform or 0.01 percent sodium azide to
prevent bacterial contamination.
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Bacteriophage Erh 1 is representative of the
Erwinia herbicola-specific bacteriophages and has been
deposited with the American Type Culture Collection,
12301 Parklawn Drive, Rockville, Maryland 20852, where
it is freely available under its accession number ATCC
8366-B. In addition to the properties described
above, Erh 1 has the following characteristics: The
head structure is transparent, flexible, and can be
twisted or flattened by various treatments. One major
protein and five minor proteins have been identified
as phage components. The genome of the phage is
estimated as having a molecular size of about 21 x
106 daltons, equivalent Jo 31 x 103 base pairs.
The bacteriophage may suitably be propagated at 23~C
either in broth or agar overlay in a medium containing
5 grams tryptone, 2 grams yeast extract, and 25 grams
glycerol per litre of water. While the bacteriophage
is stable at temperatures as high as 28C, it is
labile at higher temperatures, losing 20 percent of
its activity in 30 minutes at 37C, and 90 percent o
its activity in 30 minutes at 44C.
In a similar manner Erwinia herbicola-specific
Bacteriophages Erh 2, Erh 3, Erh 4, Erh 5, Erh 6, and
Erh 7 were also isolated from grass clippings.
Example 2
Bacteriophages Pss 8 and Pss 10, which are
representative of the Pseudomonas svringae-specific
bacteriophages, were isolated from tree litter by
phage enrichment cultures according to the procedure
of Example 1. The host strain used was Pseudomonas
svrinqae strain C-9, which has been deposited with the
American Type Culture Collection, and is freely
available under its accession number ATCC 39254. Any
Pseudomonas syringae ( c Pseudomonas syrin~ae
strain , ATCC 11043) can be substituted.
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Bacteriophages Pss 8 and Pss 10 are DNA phagesl
somewhat smaller than E. coli bacteriophage T4, with
regular icosohedral heads, contractiLe tails, T4-like
baseplates and long tail fibers. These bacteriophages
have been deposited with the American Type Culture
Collection and are available under the accession
numbers ATCC 39254-BI (for Pss 8~ and ATCC 39254-B2
for Pss 10).
In a similar manner Pseudomonas syrinqae-specific
Bacteriophages Pss 1, Pss 2, Pss 3, Pus 4, Pss 5, Pss
6, Pss 7, and Pss 9 were also isolated from tree
debris.
Example 3
The ice nucleation-inhibiting properties of the
purified Bacteriophage Erh 1 prepared in accordance
with Example 1, when added to a culture of its host
species of ice-nucleating bacteria, were determined by
means of the freezing drop method described in detail
above. At 40 minutes after introduction of the
bacteriophage to the bacteria culture, the
bacteriophage began to inhibit the ability of the
bacteria to induce ice. At 120 minutes after
bacteriophage introduction, an ice nucleation
inhibition of -3.5C was observed. At 100 minutes
after bacteriophage introduction, the ice-nucleating
bacteria were killed by bursting open to release
additional phage that had grown inside them. These
new releases of phage go on to infect more of the
ice-nucleating bacteria, until all available hosts are
infected and so deactivated. In just two hours time,
this representative bacteriophage was able to reduce a
healthy population of ice-nucleating bacteria by 90~.
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Example 4
The ability of representative Bacteriophage Erh
1, purified in accordance with Example 1, to control
frost damage on living plants was tested by the
following procedure:
Corn plants (Zea mays) were grown in plastic pots
in a greenhouse until the four-leaf stage. The pots
were randomly divided into treatment groups of up to
100 plants (400 leaves) per test. The plants in some
pots were sprayed with solutions of ~rwinia herbicola
in phosphate buffer, others with buffer alone, and
some with Bacteriophage Erh 1. These treatments all
served as controls. The test treatment plants were
sprayed to wetting with solutions of Erwinia herbicola
in concentrations of 108 bacteria cells per ml of
solution Iconcentrations greater than wound in
nature), then allowed to stabilize for 24 hours prior
to addition of 8acteriophage Erh 1 at a concentration
of 199 phage particles per ml of liquid. These
plants were allowed to stabilize prior to being
exposed to a freezing stress of -lO~C along with the
control plants.
Frost damage to the plants was quantified 24
hours after removal of the plants from the cold
chamber. Frost damage was expressed as the fraction
of the leaves per plant which exhibited frost injury
as determined by flaccid, discolored leaves. A single
area or spot of frost on a leaf classified that leaf
as being frost damaged.
The results from these experiments showed that
the plants treated with Erwinia herbicola alone
suf'ered greater than 95~ frost damage, whereas the
buffer and bacteriophage controls exhibited no
statistically significant frost damage. The plants
treated first with Erwinia herbicola, and then
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Bacteriophage Erh 1, sustained 20-25~ less damage at
-10C than the plants treated with Erwinia herbicola
alone. These results can he extrapolated to a much
higher degree of protection at milder temperatures.
At -5C it is estimated that the Bacteriophage Erh 1
will reduce frost damage due to Erwinia herbicola by
about 90%.
Since the populations of ice~forming Erwinia
herbicola bacteria on the jest plants were
substantially higher than those normally observed in
nature, and the concentration of sacteriophage Erh 1
was substantially lower than can be easily obtained in
a treatment, it is believed likely that an even
greater reduction in frost damage would be obtainable
under less severe test conditions.
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