Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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PHIP/384819
BIOLOGICAL CONTROL OF PYTHIUM DISEASE IN CROPS
Field of the Invention
[001] The invention relates to control of crop disease caused by the fungus
Fythium,
and compositions and methods therefore. In particular, the invention relates
to the
preparation and application of biocontrol agents from novel strains of
Rhizobium
leguminosarum biovar vieeae affective in controlling Pythium disease.
Background of the Invention
[002] "Damping-off' is the sudden plant death in the seedling stage due to the
attack
of fungal pathogens such as Pythium spp. and Rhizoctonia solani. The pathogens
are
soilborne and are stimulated to grow and infect the seed or seedling of host
crops by nutrients
released from a germinating seed. Damping-off disease of seedlings occurs in
most soils,
temperate and tropical climates, and in greenhouses. The disease affects seeds
and seedlings
of various crops grown under greenhouse and/or field conditions. The amount of
damage the
disease causes to seedlings depends on the fixngus, host
tolerance/susceptibility soil moisture,
and temperature. Normally; however, cool wet soils favor development of the
disease caused
by Pythium spp.. Roots may rot, or the hypocotyls (lower stem) may either
collapse or
become wiry. Seedlings may die before or after they emerge from the soil (pre-
emergence
and post-emergence damping-off, respectively). Seedlings in seedbeds often are
completely
destroyed by damping-off, or they die after transplanting. Severe loses of
plants due to pre-
and post-emergence damping-off often results in poor stands of many crops.
[003] Pythium spp. are the causal agents of seed, root, and crown rot diseases
of
economically important crops worldwide. Pythium sp. "group G", a sterile form
of Pythium
ultimum Trow, is a major plant pathogen of numerous crops grown in southern
Alberta,
including sugar beet (Beta vulgaris L.) and field pea (Pisum sativum L.).
Indoor experiments,
using soil artificially inoculated with Pythium sp. "group G", showed that
safflower
(Carthamus tinctorius L.), canola (Brassica raga L.), field pea and sugar beet
are highly
susceptible to the pathogen (Huang et al 1992). Field surveys showed that
Pythium spp. were
the main cause of poor stands of sugar beet in southern Alberta (Bardin and
Huang 2001 ).
Pythium ultimum Trow and Pythium irregulare Buisman were the principal
pathogens
causing seed rot and damping-off of field pea and reduced seedling
establishment in the
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northern Canadian prairies (Hwang and Chang 1989). Pythium diseases in field
crops are
usually controlled by seed treatment with fungicides such as ThiramTM 75 WP
and (or)
ApronTM:
[004J Increased health and environmental concerns with 'the use of chemical
fungicides have stimulated the search for alternative ways to control the
disease using
antagonistic microorganisms as biological control agents. Considerable
research has been
conducted on biological control of Pythium species using antagonistic bacteria
and fungi
(Martin and Lopes 1999). Satisfactory biocontrol of Pythium damping-off has
been achieved
using seed treatment with rhizobacteria that are antagonistic to the pathogen
(Bardin et al.
2003).
[005] Rhizobium spp. are soilborne bacteria that can establish a symbiotic
relationship with legume plants. The symbiosis takes place in plant root
nodules, in which
the differentiated rhizobia known as bacteraids convert atmospheric nitrogen
to a nitrogenous
compound that can be used by the plant. Rhizobium species are host specific.
For instance,
Rhizobiurrz leguminosarum bv. viceae Frank modulates only plants from the
genera Pisum;
Lens; Yicia, and Lathyrus. Inoculation of legume seeds with Rhizobium prior to
planting is
commonly used to improve legume crop production by increasing modulation,
thereby
reducing the need for application of nitrogen fertilizer (Brockwell et al.
1995).
[006] Several reports have indicated that Rhizobium and BradyYhizobium have
potential as biocontrol agents of plant pathogens: Rhizobia inhibited mycelial
growth of
plant pathogens such as Aphanomyces euteiehes, Phoma medicaginis (Dileep Kumar
et al.
2001), Macrophomina phaseolina; Rhizoctonia solani (Oman and Abd-Alla 1998),
Phytophthora cactorum (Drapeau et al: 1973), Fusartum spp. (Drapeau et aI.
1973; Omar
and Abd-Alla 1998; Dileep Kumar et al. 2001), and P. ultimum (Uzkoc and
Deliveli 2001).
In addition to in vitro inhibition, some Rhizobium strains reduced disease
severity caused by
Phytophthora clandestina (Simpfendorfer et al. 1999), as well as Fusarium
solani, M.
phaseolina, and Rhizoctonia solani (Siddiqui et al: 2000), in greenhouse
experiments in
which soil was artificially infested with the pathogen: In other studies,
Rhizobium
inoculation effectively suppressed diseases caused by F. sodani (Estevez de
Jensen et al.
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PHIP/384819
2002); Fusarium oxysporum, Rhizoctonia bataticola, and Pythium sp. (Nautiyal
1997) in soil
naturally infested with these pathogens.
[007] What is needed are biocontrol agents far Pythium spp.; particularly
biocontrol
agents that will protect the crops fram disease caused by Pythium ultimum.
Summary of he Invention
[008] According to the present invention, strains of the nitrogen-fixing
bacteria
Rhizobium leguminosarum biovar viceae are utilized to control Pythium
infection on crops.
The invention relates in particular to microbial pure cultures of four such
strains, identified
herein as R5, R12, R20 and R21, whichwere deposited on July 20, 2004 with the
International Depository Authority of Canada (IDAC), 1015 Arlington Street,
Winnipeg;
Manitoba, R3E 3R2, Canada, under the auspices of the Budapest Treaty, under
the following
IDAC Deposit Accession numbers:
R5: IDAC 200704-04820: 1DAC 200704-02;
812: IDAC 200704-03; 821: 1DAC 200704-01.
[009] According to one embodiment, the invention provides an antifungal
composition comprising bacteria of at least one isolated Rhizobium
leguminosarum biovar
viceae strain effective in inhibiting growth of Pythium ultimum.
[010] According to another embodiment, the invention is directed to a method
for
treating or protecting a susceptible plant from Pythium ultimum. The plant or
part thereof, or
soil surrounding the plant, is contacted with an effective amount of at least
one Rhizobium
leguminosarum biovar viceae strain which has suppressive activity against
Pythium ultimum.
[011] The bacterial strains may be elected-on the basis of their ability to
inhibit the
colonization of Pythium ultimum on an agar plate. When a bacterial strain is
said to "inhibit
the colonization of Pythium ultimum on an agar plate" means that no mycelial
growth of the
fungus occurs on a streak of the bacterial strain laid down four centimeters
distant from a
Pythium ultimum-colonized potato dextrose agar plug on the plate, following
incubation of
the plate at room temperature for five days.' The assay technique is
describedin more detail
below.
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[012] According to preferred embodiments of the invention, the Rhizobium
leguminosarum biovar viceae strain is effective in inhibiting growth of
Pythium sp. "group
G", a sterile form of Pythium ultimum, and the strains are selected on the
basis of their ability
to inhibit the colonization of Pythium sp. "group G" on an agar plate. Plants
susceptible to
Pythium sp. "group G" are treated or protected.
[013] As used in the specification and claims, the singular form "a," "an" and
"the"
include plural references unless the context clearly dictates otherwise. For
example, the term
"a cell" includes a plurality of cells, including mixtures thereof.
[014] '°Antifungal" means the ability to inhibit the growth of or kill
fungi. It should
be noted that a biological control agent can act in an antifungal manner by
not only exerting a
direct effect on a fungal pathogen; but also in an indirect manner, such as by
competing with
the pathogen for nutrient. Both such direct and indirect actions are
understood to be
"antifungal".
[015] As used herein, "biovar" or "biological variant" (or the abbreviation
"bv.")
means a strain of a bacterium that is differentiated. by biochemical or other
non-serological
means from another strain. A "strain" is a subset of bacterial species
differing from other
bacteria of the same species by some minor but identifiable difference.
[016] As used herein, "biological control" is defined as control of a pathogen
or
insect by the use of a second organism.
[017) As used herein, the term '"comprising" is intended to mean that the
compositions and methods include the recited elements, but not excluding
others.
[018] The term "culturing" refers to the propagation of organisms on or in
media of
various kinds.
[019] A "composition" is intended to mean a combination of active agent and
another compound or composition, inert (for example, a detectable agent or
label) or active,
such as an adjuvant.
[020] An "effective amount" is an amount su~cient to effect beneficial or
desired
results: An effective amount can be applied in one or more applications. In
terms of
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treatment and protection, an "effective amount" is an amount sufficient to
ameliorate,
stabilize, reverse, slow or delay progression of a fungal infection.
[021] An "isolate" is a pure culture derived from a heterogeneous, wild
population
of microorganisms.
(022] 'The term "isolated" is used interchangeably with "biologically pure"
and
means separated from constituents; cellular and otherwise, in which the strain
or metabolite is
normally associated with in nature.
[023] As used herein, "Pythium ulfimum" is meant to include all forms of the
species of this name, including but not limited to Rythium sp: "group G".
[024] By "suppressive activity" of a biological control;agent against a fungal
pathogen is meant the ability of the agent to ameliorate, stabilize, reverse,
slow or delay
progression of an infection by the fungal pathogen.
[025] "Whole broth culture" refers to a liquid culture containing both cells
and
media.
Detailed Description of the Invention
[026] We have found that Pythium diseases may be controlled by strains of the
nitrogen-fixing bacteria Rhizobium leguminosarum bv. viceae. In one study,
fifty-six,percent
of strains of R: leguminosarum bv. viceae obtained from field pea and lentil
nodules were
found to improve emergence of sugar beet seedlings in soil artificially
infested with the
pathogen Pythium sp. "group G" in indoor experiments. Strains tested as seed
treatments in
diseased fields affectively controlled damping-off of sugar beet and field pea
caused by
Pythium spp. The effectiveness ofthe Rhizobium strains is similar to that
ofPseudomonas
fluorescens Migula LRC 708, a biological control agent against Pythiz~m
damping-off of
sugar beet, field pea, canola, and safflower (Bardin et al. 2003): The
biological control
activity of the Rhizobizeni strains is not host dependent, as they are
effective agents for both
legume (pea) and nonlegume (sugar beet) plants.
[027] According to the present invention; R, leguminosarum bv, viceue strains
are
utilized to control or alleviate Pythium infection on crops. Rhizobium
leguminosarum bv.
viceae strains are useful for promoting plant health by protecting inoculated
seeds from attack
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by Pythium ultimum, and Pythium sp. "group G" in particular; thereby reducing
incidence of
damping-off diseases.
(028]' The bacteria Rhizobiurn leguminosaxum bv: viceae comprising the active
agent
ofthe invention may be isolated tom riot nodttl~s of host--'xgutnes such as
pea and lentil:
The bacteria are collected from root nodules which are removed from the plants
and crushed.
The nodule contents are plated on an appropriate medium to support the growth
of the
bacteria. The: bacteria are cultured under conditions favoring the growth
thereof. Conditions
for culturing Rhizobium leguminosarum strains are known to those skilled in
the art, and are
exemplified in he Examples which follow. Colonies are isolated from the
culture.
(029] The antagonistic activity of isolates against Pythium ultimum may be
determined using the dual-culture technique. Each bacterial strain is streaked
on a medium
which will support the growth of Pythium ultimum; e.g., Tryptone Yeast Extract
agar near the
edge of a Petri dish (9 cm diameter). After incubation for one day at room
temperature, a
rnycelial plug (0.6 mm diameter) of Pythium ultimum-from a 48-hour culture
grown on potato
dextrose agar is placed in the center of each dish containing the bacterial
streaks. The plates
are incubated for five days at room temperature (20 t 2 °C) and the
inhibitory effect of each
bacterial strain is determined by measuring ;the inhibition zone t~f mycelial
growth. The
inhibitory effect is scored as positive where the Pythium growth stops on ar
before the
bacterial streak line.
[030] The bacteria may be utilized in the form ofcultures of bacteria, such as
a
suspension in a whole broth culture, to prepare appropriate compositions for
ground
treatment, plant treatment, soil and/ or growing media reatment, or seed
treatment.
(031] The compositions containing the bacteria as the sole active ingredient,
or as a
combination with one or more other active ingredients, are prepared in known
manner, such
as by using standard fermentation methods, .processes and equipment, followed
by
homogeneously mixing and/or grinding the active ingredients with extenders;
growth media
ingredients (for example such as nutrients, stabilizers; buffering systems,
plant growth
hormones; and pH adjustment ingredients) and liquid or dry organic or
inorganic carriers.
Suitable carriers include sterilized and sanitized liquid earners; pre-
sterilized (irradiated or
steam sterilized) and non-sterilized peat powders; granulated, spheronized or
pelletized peat,
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clay; and other extenders, filler pigments or minerals. Other carriers for
granular formation
include talc, gypsum, kaolin, attapulgite, montmorillonite; bentonite; wood
flour, ground corn
cob grits, starch, cellulose, and bran. The formulations can also contain
additives such as
adhesives, stickers, binders, polymers and other adjuvants applicable to
agricultural or
horticultural applications: Stickers or binders may comprise, for example,
ethylene glycol,
mineral oil, polypropylene glycol, polyvinylacetate, lignosulfonate, polyvinyl
alcohol,
polyvinylpyrrolidone, graphite, gum Arabic, methyl cellulose, and sucrose.
[032] The compositions of this invention can be formulated in powder or
granular
form by mixing together all the components, including any carrier and/or other
additives)
which may be utilized until a homogeneous: mixture is formed. A sticker, if
employed, may
then be added and the entire mass mixed again until ithas become essentially
uniform in
composition. The composition may or may not be formulated in a pre-sterilized
carrier
system.
[033] The optimum concentration of Rhizobium leguminosarum bv. viceae
employed in the compositions of the invention for a particular application can
be readily
determined by those skilled in the art. In general, the concentration of
bacteria can range
from about 0.001 to about 1 %, preferably from about 0:01 to about 0.5%, more
preferably
from about 0.05 to about 0.1%, by weight.
[034] In the case of a liquid formulation; an aqueous liquid nutrient medium
may be
utilized, optionally comprising adjuvants such as stickers; stabilizers and
colorants.
[035] The composition of the invention may contain, as additional active
agents,
cells, spores or propagules of other biological control agents, one or more
chemical
fungicides, or one or more other pesticidal materials, such as insecticides.
The chemical
fungicide may be selected on the basis of its activity against Pythium spp.,
or may be selected
on the basis of activity against other fungal pathogens.
[036] The method of the invention comprises applying to plants an antifungal
effective amount of a composition containing Rhizobium leguminosarum bv.
viceae. The
composition is most advantageously applied to roots or seeds. The composition
may also be
utilized as ground treatments in fields or greenhouses. They may be applied to
soil
surrounding plants, or applied to soil into which seeds or seedlings are
planted.
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[037] The compositions are applied by methods which include, for example; seed
treatments, spray applications, in-furrow applications; soil and growing media
inoculation,
application through irrigation system, and the like. The comp4sitions can be
applied as stand
alone or with the standard chemical treatments to control Pythium.
[038] When employed as a seed dressing, the amount of composition is applied
such
that the seed is coated with a concentration of bacteria adequate to provide
protection against
Pythium spp. The actual amount to utilize depends on the nature and size of
the seed, the
amount of the protection desired, the local soil conditions, and other factors
which may be
taken into account in selecting the appropriate dosage of bacteria.
Appropriate application
rates of bacteria in terms of colony forming units (cfu) per seed are as
follows: large seeded
crops - from about 104 to about 10$ for legumes, and from about 106 to about
10~ for non-
legumes; medium size-seeded crops - from' about 103 to about 10' for legumes,
and from
about I05 to about 106 for non-legumes; small seeded craps - .from about 1 O2
to about 106 for
legumes; and from about 104 to about 105 for non-legumes. Greater
concentrations of
bacteria may be applied.
[039)- Seeds may be bacterized according to the present invention by steeping
the
seeds in a suspension of bacteria for an appropriate time, e:g. one hour.
According to one
such technique, bacterial slurries are prepared by adding 3 ml of 1 % methyl
cellulose to
tryptone yeast medium plates on which the 'culture is grown, and gently
scraping the culture
off the plate. For seed treatment, seeds are aoaked for, e.g., 20 minutes in
the bacterial slurry.
The bacterial concentration in the slurry may be-determined by plating serial
dilutions on
tryptone yeast medium (Beringer, 1974).
[040] According to one embodiment, an inoculant composition maybe prepared for
application to seeds, using ground peat. Methods for the preparation of
inoculant
compositions of Rhizobium sp. for inoculation of crops; e.g., legumes, to
increase nitrogen
fixation are known. See, e.g., U.S; Pat. 5,4$4,464, the entire disclosure of
which is
incorporated herein by reference. One such inoculant composition is prepared
from sterilized
powdered peat with a moisture content of 6-20%, with or without a sticker.
Using aseptic
techniques, a suspension ofRhizobium leguminosarum bv. viceae is added to the
peat at a
rate of from about 105 to about 10g colony forming units of bacteria per gram
of peat.
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[041] The composition may be utilized to protect any drop which is susceptible
to
infection and damage by Pythium ultimum, and Pythium sp. "group G" in
particular. Such
crop species include, for example; sugar beet {Beta vulgaris L.), field pea
(Pisum sativum L:),
lentil (Lens spp.), safflower (Carthamus tinctoYius L.), canola {Brczssica
raps L. and Brassica
napus L.), chickpea (Cicer spp.), sunflower (Helianthus spp.), alfalfa
(Medicago spp:), .
soybean (Glycine spp.), and field bean (~cia faba).
Examines
[042] In the following Examples; llle viable counts of bacterial agents in
slurries and
on seeds were expressed as mean cfu t SE. Shoot dry mass and emergence data of
both
indoor and field experiments were analyzed tatistically using the Statistic
Analysis Software
package Version 6Ø9 (Examples 1-6) or Version 8.2 (Example 7) (SAS Institute
Inc:, Cary;
N.C.). Analysis of variance was done using the general linear rrlodel
procedure. Differences
between treatments were analyzed using Fisher's least significant difference
(LSD) test. All
analyses were performed at the P = 0.05 level.
Example 1
Isolation of Rhizobium Strains
(043] Strains of R. leguminosarum bv. viceae were isolated from root nodules
of
field pea and lentil grown in southern Alberta, Canada, as follows: Roots from
twa plants per
crop were washed in water to remove soil particles. The nodules were excised,
surface
sterilized in 2% sodium hypochlorite for l min, washed eight times in sterile
distilled water,
and crushed with a sterile spatula in 200 ~L sterile water. The nodule
contents were plated
on tryptone - yeast extract medium {TYBeringer 1974) containing 1.5% agar
(Difco,
Detroit, Mich:). Following incubation for 3-4 days at room temperature (20 t 2
°C), a
colony from each plate was purified by three successive single colony
isolations. Eigllteen
strains of R. leguminosarum bv, viceae were isolated in this manner. Ten
strains were
isolated from field pea root nodules and eight from lentil root nodules. Of
these strains, 8
showed no potential for control of Pythium damping-off of sugar beet in
preliminary indoor
experiments and were not tested further. The identities 6f the l 0 remaining
strains, 8 from
pea and 2 from lentil (Table l ), were confirmed by performing plant
nodulation experiments
(see below) and by streaking the bacteria on Luria-Bertani (LB, Miller 1972)
agar. The
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strains did not grow on LB; which is consistent with the fact that 1Z
deguminosarum is
sensitive to the high salt concentration contained in this media.
Ezample 2:
Plant Nodulation by RhiZObium Strains
j044] The ability of the ten Pythium-antagonizing Rhizobium isolates to form
nitrogen-fixing nodules on pea and lentil plants was determined in a nitrogen-
free medium.
Seeds were surface sterilized for 5 min in 50% aqueous sodium hypochlorite,
washed 8-10
times with sterile distilled water, and germinated fort days in the dark on
water agar (1.5%)
in Petri dishes. Six seeds were planted in each sterile Leonard jar assembly
{Leonard 1943),
containing a mixture of quartz sand and vermiculite (l :l; v/v) saturated with
nitrogen-free
Jensen's nutrient solution (Vincent 1970). Two days after planting the seeds,
each jar was
inoculated with 10 mL of an aqueous bacterial suspension (107-108 cfu/10 mL)
ofRhizobium
or with 10 mL water far the uninoculated control. Each treatment was:
performed in
duplicate: The experiment was repeated once. The plants were kept in a growth
cabinet in a
16 h light (20 °C): 8 h dark (I5 °C) cycle. They were watered
with sterile distilled water as
required. Lentil and pea.plants were collected 26 and 27 days after
inoculation; respectively.
The shoots of the plants were excised, dried in a 60°C oven for 5 days;
and weighed to
determine nodulation efficacy.
[045] Plants inoculated with each of the ten Pythium-antagonizing Rhizobium
strains
were green and healthy compared with the brown and stunted plants of the
uninoculated
control. There were pinkish nodules formed on the roots of plants inoculated
with the strains,
while no nodules developed on the roots ofuninoculated plants: In addition,
the dry shoot
masses of the inoculated plants were significantly(P < 0.05) greater than
those of
uninoculated plants (Table 1). Strain Rl2 was effective in establishing a
beneficial symbiotic
interaction with both lentil and pea plants (Table 1 ).
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Table 1: Source of RhiZobium leguminosarum bv. viceae strains and their
nodulation efficacy on
i;eld pea and lentil.
Rhizobium Shoot
dry
mass
leguminosarum S %
control
)
bv: viceae* Plant source"i'Pea Lentil
Strain
R3 Pisum sativum245a~ nd
R4 Pisum sativum243a nd
R5 Pisum sativum295a$ nd
R7 Pisum sativum288a~ nd
R8 Pisum sativum263a$ nd
R9 Pisurn sativum294a~ nd ,
R12 Lens culinaris273a~ 306a'[
R1.9 Lens culinarisnd 327a'[
R20 Pisum scativum235a nd
R21 Pisum sativum224a nd
Uninoculated control 100b IOOb
Note: Nodulation efficacy is expressed as percent increase in shoot dry mass
of a pea or lentil planE inoculated
with a R, leguminosarum bv, viceae strain compared with the un'tnoculated
control {100%)., 'The values
represent the means of 12 plants (two pots of 6 plants each? from two
independent experiments. Means within
the same column followed by the same letter are notsignificantly different at
P = 0.05 level (Fisher's LSD test).
nd, not determined.
*Strains of R. Teguminosarum bv. viceae isolated from the root nodules of pea
or lentil plants collected in
southern Alberta.
Plant nodules where the bacteria were isolated.
$Dry shoot mass of the uninoculated control was 202.3 mg/plant.
~Dry shoot mass of the uninoculated control was 271.0 mg/plant.
!Dry shoot mass of the uninoculated control was 83.0 mg/plant.
Ezample 3
Control of Pythium Damping-off of Sugar Beet by Rhizobium Strains
(dual culture experiments)
[046] The antagonistic activity of the ten remaining R. leguminosarum bv:
viceae
strains against Pythium sp. "group G" strain LRC 2105 (Huang et al. 1992) was
determined
by streaking a Rhizobium strain 4 cm away from a potato dextrose agar (PDA)
plug colonized
by Pythium on TY agar plates (dual culture technique). After incubation at
room temperature
for 5 days, the inhibitory activity of the Rhizobium strain was determined by
measuring the
zone of mycelial growth inhibition around the bacterial streak. Three ratings
were used: -, no
inhibition zone and growth of Pythium over the bacterial streak; +, no
inhibition zone, but no
growth of Pythium on the bacteria streak; and ++, 1-5 mm inhibition zone.
There were three
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replicates for each treatment and the experiment was repeated once. Strain RS
was rated as
++: It was the only strain showing: antagonistic :effects to Pythium sp.
"group G", with
formation of a small zone of inhibition 2 mm in size. The other 9 strains did
not exhibit
zones of inhibition but were able to prevent colonization of the bacterial
streak by the
pathogen and were therefore rated as + (slight inhibition).
Example 4
Test for Protease Production by Rhizabium Sfrains
[047] Protease production was determined by incubating colonies of R:
leguminosarum bv. viceae on skim milk agar plates (Dunne et al. 1997) for 5
days at room
temperature (20 t 2 °C). Protease activity was compared with ';the
protease positive strain,
Pseudomonas fluorescens Migula LRC 708; which degrades casein and causes
clearing of the
skim milk agar plate (Bardin et al. 2003). There were three replicates for
each treatment and
the experiment was repeated once. Unlike strain P. fluorescens 708, none of
the Rhiaabium
strains tested showed protease activity, as they failed to produce clearing
zones around the
colonies when plated on the skim milk agar plates. Thus, production of
extracellular
proteases is not the mechanism of action of the Rhiaobium strains.
Ezample 5
Seed Treatment by RhizoUtum Strains (indoor experiments)
[048] The strains of R. leguminosarum bv. viceae were further tested as seed
treatments for control of Pythium damping-off of sugar beet in nonsterile
soil. Bacterial
cultures were grown on TY agar in Petri dishes (5.5 cm in diameter) for 48
hours at room
temperature. The bacterial culture was resuspended in 3 mL of 1 % methyl
cellulose (MC)
(Aldrich Chemical, Milwaukee, Wis,) by scraping the agar surface gently with a
spatula.
This resulted in bacterial slurries with a concentration averaging 3.9 x 109 t
0.5 x 109 {mean
f SE) cfu/mL. Sugar beet {Beta vulgaris 'HM Bergen') (Novartis Seeds -
Hilleshog;
Longmont, Colo.) seeds were soaked for 2Q minutes in the MC-bacterial slurry
and were
seeded directly into soil artificially infested with Pythium sp. "group G"
strain LRC 2105.
The soil consisted of 3 parts topsoil {Bzdell Soil Service, Lethbridge,
Alberta.), 1 part sand
{Tollestrup Construction, Lethbridge, Alberta); and 1 part peat moss (Premier
Horticulture,
Red Hill, PA:). The Pythium inoculant was prepared in pans containing a
sterile mixture of
150 g wheat bran {Ellison Milling, Lethbridge; Alberta), 150 g corn meal
(McCormick,
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London, Ontario), and 300 mL distilled water. Twenty plugs (8 mm in diameter)
of a 48-
hour-old PDA culture of Pythium sp. "group G" were placed in each pan. After
incubation
for 2 weeks at room temperature in the dark; the wheat bran - corn meal mix
was completely
colonized by the pathogen. The Pythium inoculum was air-dried at room
temperature for 4
days and ground using a Thomas-Wiley model 4 Laboratory mill (Thomas
Scientific,
Philadelphia, Pa.) equipped with a 1-mm mesh screen. The soil, artificially
infested with
Pythium sp. "group G" at a concentration of 2 g inoculum/kg soil, was used to
fill root
trainers (Spencer-Lemaire Industries, Edmonton, Alta.), each containing 17
books of six cells
per book. One sugar beet seed was planted per root trainer cell at a depth of
1.5 cm.
Uninoculated seeds were also planted in non-infested soil. The root trainers
were soaked in a
water-filled tray until the soil was saturated by capillary action, and were
then placed in
propagator trays (The Stewart Company, Croydon, Surrey, UK) to create a high-
moisture
environment. The propagator trays were kept in a growth chamber in a 16 hour
light (20 °C):
8 hour dark ( 15 °C) cycle. In each experiment here were three
replicates per treatment and
18 seeds per replicate. The treatments were arranged in a completely
randomized design.
Seedling emergence was recorded 14 days after planting, and data .from
bacterial eed
treatments were compared with the uninoculated control. Each set of
experiments was
repeated twice. Non-germinated seeds were collected, washed with sterile
water, surface
sterilized in 70% ethanol for 2 min, and plated on PDA in Petri dishes. The
fungi isolated
from the seeds were purified on PDA; and thegenus pf each fungus isolated was
determined
based on morphological characteristics.
[049] Emergence of uncoated sugar beet seeds planted in the Pythium-infested
soil
used in the indoor experiment was reduced by 37% (21 % emergence) compared
with seeds
planted in non-infested soil (58% emergence). Pythium was reisolated from 65%
of the non-
germinated seeds tested. Despite the lack of clear antagonism against Pythium
sp: "group G"
in the in vitro assays, seed treatment with the Rhizobium strains
significantly (P < 0.05)
increased emergence of sugar beet in soil artificially infested with Pythium
sp: "group G"
compared with the untreated control (Table 2). The most effective strains for
biological
control of damping-off of sugar beet were R3, R4; R5, R7, R12, R20; and R21
(Table 2).
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Table 2: Control of Pythium damping-off of sugar beet (Beta vulgaris) by seed
treatment with
R. teguminosarum bv. viceae (indoor experiments):
Rhizobium leguminosarum
bv. viceae
Strain Emer"g-ence ~%)
R12 52a
R20 46ab
g21 44ab
R4 43abc
R3 42abc
R7 42abc
RS 4l abc
R9 39bcd
R8 36bcd
Rl9 32ed
Untreated control 2 i a
Note: Emergence of sugar beet seedlings was detenriined 14 d after planting:
Means are of three replicates from
three independent experiments: All experiments gave similar results. Means
followed by the same letter are not
significantly different at P = O.US. level (Fisher's LSD test):
Example 6:
Control of Pythium Damping-off of Sugar Beet and Field
Pea by Rhizobium Legumitrosarum bv: Vieeae Strains (field experiments)
(050] The selected strains ofR.-le~uminosarum bv. viceae (R12, R20, and R21)
effective against Pythium damping-off of sugar beet in indoor experiments were
tested for
control of damping-off of sugar beet and field pea in fields naturally
infested with Pythium
spp. at the Lethbridge Research Centre; Alberta. The efficacy ofthe Rhizobium
strains was
compared with the biocontrol agent P. fluorescens 708, which was shown to
improve
emergence of sugar beet; field pea, canolaand safflower in soil naturally
infested with
Pythium spp. (Bardin et al. 2003). The seeds were coated with the bacterial
slurry as
described previously using 2:4 and 9.S mL bacterial slurryllOU seeds of sugar
beet and f eld
pea, respectively. The seeds were dried overnight at room temperature on a
metallic mesh,
which was placed on a paper towel to absorb the excess slurry. The number of
bacteria
coated onto the seeds was similar for the four bacterial strains, ranging from
1.4 X 106 ~ 0.2 x
106 to 2.3 .x 10' ~ 0.2 x l 0' cfulseed for sugar beets and 3.0 x l 0' ~ 0.3 X
10' to 1.2 X l Os ~
0.4 X 10$ cfu/seed for field peas. The coated seeds were then stored at 4
°C until planting.
Bacterial counts on the seeds were deternnined by vortexing fve coated seeds
in 5 mL of
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distilled sterile water for 30 seconds, and by plating serial dilutions on T1'
agar medium in
Petri dishes for Rhizobium strains and on PI7A in Petri dishes fox P.
fluorescerrs. Each
bacterial count was performed in duplicate, and bacterial determinations for
each treatment
were performed twice. The Rhizobiurra-treated and untreated seeds were machine
seeded into
0.9 m wide x 5.0 m long plots made of 4 rows of 100 seeds/row in a field
naturally infested
with Pythium spp. The plots were trimmed to 3.5 m after all seedlings emerged.
Treatments
were arranged in a randomized complete block design, with six replicates per
treatment. The
field experiments were performed twice, once in May and again in August 2001
in Fairfield
Farm, Lethbridge, Alberta. Seedling emergence was recorded 4 weeks after
planting and was
compared with the uninoculated and fungicide controls. The amount of Thiram~
for the
fungicide-treated seeds was 90 g/25 kg sugar beet seeds and 30 g/25 kg field
pea seeds.
[051] Treatment of pea seeds with R. leguminosarum bv. viceae strain R12 or
R20
caused a significant (P < 0.05) increase in seedling emergence compared with
the untreated
control in the two field experiments (Table 3). The efficacy of the two
Rhizobium strains was
similar to that of seed treatments with the rhizobacteriurn P. fluorescens
708. Rhizobium
leguminosarum bv. viceae R21 significantly increased pea seedling emergence
compared
with the untreated control in the second (August 2001) but not in the first
(May 2001) field
experiment. The level of seedling emergence in the second field experiment was
lower but
not significantly (P > 0.05) different from that of R. leguminosarum bv.
viceae R20 and P:
fluorescens 708. None of the bacterial treatments were as effective as the
fungicide
ThiramTM for control of damping-off of field peas.
[052J_ In the sugar beet experiments conducted in May and August of 2001, seed
treatment with R. leguminosarum bv. viceae R12, R20, or R21 increased seedling
emergence
compared with the untreated control (Table 3): This increase was significant
(P < 0.05) in the
August experiment. In both the May and August field experiments, the percent
emergence of
the Rhizobium-treated seeds was not significantly different from that of seeds
treated with P.
fluorescens 708. Rhizobium leguminosarura bv. viceae R12 and R21 were as
effective as the
fungicide treatment for protection of sugar beet seedlings against Pythium
damping-off in the
August field experiment, while P. fluorescens 708 was as effective as the
fungicide treatment
in the May field experiment (Table 3):
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PHIP/384819
Table 3: Effect of bacterial seed treatment on: field pea and sugar beet
emergence in a field
naturally infested with Pythium spp:
Emergence (%)
P~ Sugar beet
(Pisum (Beta vulgaris)
sativum)
May August May August
Treatment 2001 2001 2001 2001
Untreated control 41.4d 12.$d lO.Oc 6.5d
Fungicide (ThiramTM)~' 71.4a 48.3a 28.6a 27:0a
R12 54.3bc 34.4b 18.6bc 24.7abc
1120 51.4c 30:6bc 14.3bc 21.9c
R21 37.14 23:7c l7.Ibc 24.9ab
Pseudomonasfluorescens 60:0b 29.6bc 20.Oab 23.Sbc
708
Note: Seedling emergence
was determined 4 weeks
after planting. Values
are means of six replicates.
Means
within each column followed
by the same letter are
not significantly different
at P = 0.05 level (Fisher's
LSD
~~):
The concentration of ThiramTM
was 90 g/25 kg sugar beet
seeds and 30 g/25 kg field
peas.
Example 7:
Control of Pytlaium Damping-off of Pea and Y.entil by
Rhizobium Strains (field experiments)
[053] The strains of Rhizobium leguminosarum bv: viceae used for the study
were
99A1; R12, R20, and R21. Strain 99A1 was originated from the commercial pea
inoculant
produced by Agrium, Inc. Calgary, Alberta. Bacterial cultures were grown on
tryptone-yeast
agar (TYA) (Beringer, 1974) in Petri dishes for 48 h at zoom temperature
(2012°C). The
resulting colonies were suspended in 5 ml per dish of I% methyl cellulose
(Sigma-Aldrich,
Milwaukee, W>) in sterile distilled water, and scraped gently with a spatula
to obtain bacterial
slurries. Seeds of field pea cv. Trapper and lentil cv. Laird were soaked for
20 minutes in the
slurries, spread on a metallic mesh sheet with paper towel underneath to
absorb the excess
slurry; and air-dried overnigfit under a fume hood: Enumeration of bacteria
coated onto seeds
was done by placing 5 seeds in a test tube with ~ ml of sterile distilled
water, vortexing for 30
sec, and plating serial dilutions on TYA, 0:1 ml per 9-cm dish: After
incubation at room
temperature for 3 days, bacterial colonies developed in each dish were
counted. There were
two replicates for each treatment.
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[054] Field experiments were conducted at the Agriculture and Agri-Food Canada
Research Centre near Lethbridge, Alberta, Canada; in a field naturally
infested with Pythium
spp: (predominantly Pythium sp. 'group G'). For the pea experiment, seeds were
planted
using a plot seeder on 28 May 2004, in 4-row plots with a row length of 5 m, a
row spacing
of 22.5 cm, and a plant spacing of 5 cm' (i.e. 20 seeds/m). Untreated seeds
and fungicide-
treated seeds ('ThiramTM at the rate of 30 8125 kg,seed) (Gustafson; Calgary;
Alberta; Canada).
were used as controls. Treatments were arranged in a randomized block
designwith 6
replicates. For the lentil experiment, seeds were planted on the same date and
using the same
parameters as for field pea.
[055] Seedling emergence for each plot was determined. 'The number of healthy
seedlings and the number of wilted seedlings were counted in the middle 3 m of
each row,
and the percent loss due to pre-emergent and post-emergent damping-off were
calculated; as
well as the final stand establishment. The causal agent of seedling death was
determined by
collecting 10 non-emerged seedlinigs and ali of the wilted seedlings from each
plot; washing
in running water, surface sterilizing in 70% ethanol for 90 sec, incubating on
potato dextrose
agar (PDA) in Petri dishes at room temperature for 7 days, and examining the
organisms
derived from each sample. Results of reisolation of diseased seedlings were
used to calculate
the incidence of damping-off due to Pythium spp. for each plot.
[456] Seedling height for each plof was determined (6-node stage for peas; 5-
node
stage for lentils). For each row, ten seedlings were randomly selected and
the: distance :from
the first node to the terminal branch of each seedling was measured.
[057] Differences between treatments for incidence of damping-off, seedling
emergence and seedling height data were analyzed for statistical significance
using analysis
of Variance (ANOVA) and means of treatments for each set of data were
separated 'using
Duncan's multiple range test at the P~.OS level. 'All statistical analyses
were done using
SAS Statistical Analysis Software, Version,8:2 {SAS Institute Inc., Cary,
North Carolina
2001). The results are set forth in Tables 4 -7.
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PHIP/384819
Table 4: Effect of
seed treatment with
Rhizobium leguminosarum
biovar viceae strains
on damping-off of
field pea.
Dampin -g Final
off (doll
Treatment Pre-emer Post-erner~ent~PythiumStand (%)1
eg nt b
Control 62.0 a2 0.2 55:4 37.8 a2
a2
99A1 62.0 a 0.3 54.8 37.7 a
a
R12 55.2 ab 0.1 47.0 44.7 ab
ab
R20 51:6 b 0:7 43.4 47.9 b
b
g21 28.5 c 0.8 23.7 70.7 c
c
Fungicide {Thiram) 20:3 c 0.6 16.7 79.1 c
c
' Based on 60 seeds planted per 3-meter section of row, 4 rows per plot.
Z Means within each column followed by the same letter are not significantly
different (Duncan's
multiple range test; P~0.05):
Table 5: Effect of seed treatment with Rhizobium leguminosarum biovar viceae
strains
on seedling height of field pea.
Treatment Plant Heig it (cm)2
Control 11.l a2
R20 11.1 a
99A1 12.2 ab
R12 12:8 b
R21 12.8 b
Fungicide (Thiram) 14.6 c
' Distance from the first node to the terminal branch; measured at the 6-node
stage (4 weeks after
planting): Based on random selection of 10 seedlings per row, 4 rows per plot.
Mews within each column followed by he same letter are not significantly
different (Duncan's
multiple range test; P>0.05).
Table 6: Effect of seed treatment with Rhizobium leguminosarum biovar viceae
strains
on damping-off of lentil.
Damping-off %) Final
1
Treatment Pre-emeruent by PvthiumStand (%)1
Post-emergent
99A1 50:6 2.3 38:1 a2 47.1 a2
a2
Control 46.3 2.7 35.3 ab 51.0 ab
ab
R21 44. 1.~ 31.5 be 54.4 be
i
ab
R20 43.1 3.3 30.2 be 53.6 be
b
R12 39.9 2.2 27.8 cd 57.9 cd
be
Fungicide (Thiram)34.2 2:6 22.8 d 63.2 d
c
' Based on 60 seeds planted per 3-meter section of mw, 4 rows per plot.
Z IVleans within each column followed by the-same letter are not significantly
different (Duncan's
multiple range test; P~0.05).
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PHIP1384819
Table 7: Effect of seed treatment with Rhizobium leguminosarr~m biovar viceae
strains
on seedling height of lentil.
Treatment Plant Heigh~cm~
Control 11.2 a2
R20 11.2 a
99A 1 11.2 a
R12 11.3 a
~1 11.3 a
Fungicide (Thiram) 11.4 a
' Distance from the first node to the terminal branch; measured at the S-node
stage (4 weeks after
planting): Based on random selection of 10 seedlings per row, 4 rows per plot.
z Means within each column followed by the same letter are not significantly
different (Duncan's
multiple range test; P>O.OS).
(058] Enumeration of bacteria coated onto seeds revealed similar numbers of
bacteria per seed for all four strains of R: leguminosarum bv. viceae, for
both field pea and
lentil. The number of colony-forming units (cfu) per seed ranged from 2.3 X
105 to 2.9 X 105
for pea, and from 2.2 x 10$ to S.l x 105 for lentil.
[059] Reisolation of diseased pea seedlings revealed that 84% of the seedlings
killed
by pre- and post-emergent damping-off were infected with Pythium spp., whereas
the
remaining seedlings were colonized by Fusarium spp. Treatment of pea seeds
with R20, R21
or ThiramTM significantly (P<O.OS) reduced pre-emergent damping-off compared
to the
untreated control (Table 4). The incidences of pre-emergent damping-off for
the treatments of
R20, R21 and ThiramTM were S 1.6%, 28:5% and 20.3%, respectively, compared to
62.0% for
the untreated control. There was no significant difference in incidence of pre-
emergent
damping-off between the treatments of R21 and ThiramTM: Damping-off losses of
pea due to
Pythium spp. alone followed a similar trend; ranging from 16.7% in he
fungicide treatment
and 23:7% in the treatment of R21, to SS:4% in the untreated control (Table
4). The height of
pea plants arising from seed treated with R12, R21 or ThiramTM was
significantly (P<O.OS)
greater than for plants arising from untreated seed (Table S). Seedling height
for the
treatments of Rl2 and R21 was 12.8 cm for both Rhizobium strains; compared to
14.6 cm for
the treatment of ThiramTM, and 11.1 cm for the untreated control:
(060] For the lentil experiment, results of plating of diseased seedlings
showed that
68% were infected with Pythium spp:, 22% were infected with Botrytis cinerea,
and the
remainder was colonized by Fusarium spp. Treatment of lentil seeds with R20,
R12 or
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CA 02476531 2004-08-04
PHIP/384819
ThiramTM significantly (P<0.05) reduced incidence of pre-emergent damping-off
compared
to the untreated control (Table 6). The disease incidences for the treatments
of R20, Rl2 and
ThiramTM were 43.1%, 39.9% and 34.2%, respectively, compared to 50.6% for the
untreated
control. Incidence of damping-off of lentil due to Pythium spp. alone followed
the same
trend; ranging from 22.8% in the fungicide treatment and 27.8% in the
treatment of R12, to
38.1% in the untreated control (Table 6). No significant differences in
seedling height of
lentil were detected among the treatments (Table 7).
[061] Among the four strains of R. leguminosaYUm bv. viceae tested, strains
R20 and
R21 from pea were most effective for control of damping-off of pea (Table 4),
whereas the
strain R12 from lentil was most effective for control of damping-off of lentil
(Table '6). The
study on pea also suggests that the strains may have a growth promoting effect
on seedlings,
as seen in the case of increased height of pea seedlings for the treatments of
R12 and R21
(Table 5).
[062] All references discussed herein are incorporated by reference. One
skilled in
the art will readily appreciate that the present invention is well adapted to
carry out the
objects and obtain the ends and advantages mentioned, as well as those
inherent therein. The
present invention may be embodied in other specific forms without departing
from the spirit
or essential attributes thereof and; accordingly; reference should be made to
the appended
claims, rather than to the foregoing specification, as indicating the scope of
the invention.
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PHIP1384819
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