Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
COM~OSr~ON COM~RISLNGPLANTG~OWTHPROMOTDNG RH~OBACTERIA
.
FIELD OF THE INVENTION
The present invention relates to compositions comprising
plant growth promoting rhizobacteria (PGPR) and methods therefor for
enhancing the formation and development of root nodules in legumes, so as
to enable an increase in the growth and yield thereof under conditions that
inhibit or delay nodulation. More particularly, the invention relates to
compositions comprising PGPR and methods therefor for increasing grain
yield and protein yield of soybean grown under environmental conditions that
inhibit or delay nodulation, such as suboptimal root zone temperatures
(RZTs).
BACKGROUND OF THE INVENTION
The knowledge that elements in the soil influence root
nodulation has long been recognized. Indeed, the Romans transferred soil
from successful legume fields to unsuccessful ones in order to improve the
quality of the latter.
It has since then been den,onst,dl~d that one important soil
element responsible for nodulation is soil bacteria. The family Rhizobiaceae
consists of a heterogeneous group of gram-negative, aerobic,
non-spore-forming rods that can invade and induce a highly differentiated
structure, the nodule (on the roots, and in some instances, stems of
leguminous plants), within which atmospheric nitrogen is reduced to
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ammonia by the bacteria. The family Rhizobiaceae contains three genera,
Rhizobium, Bradyrhizobium, and Azorhizobium. The host plant is most often
of the family Leguminosae. The slow-growing nodulation bacteria which have
specific associations with soybean are referred to as Bradyrhizobium.
5 Currently, Bradyrhizobium has only one named species B. japonicum, with
others lumped together in a miscellaneous group (Barbour et al., 1992);
these latter strains are referred to as B. sp., followed by the plant species
they infect in parenthesis. Some soybean plants can also nodulate with the
fast growing Rhizobium fredii (Sprent et al., 1990). Rhizobium species,
10 sometimes designated "fast-growing" rhizobia, include among others R.
meliloti which infects alfalfa.
The element N is essential to all living organisms because
it is a component of many biologically important molecules. The most
important of these include nucleic acids, amino acids and therefore proteins,
15 and porphyrins, which occur in large amounts in all living cells. To be able to
multiply and grow, or just survive, organisms require a source of N. The
ability to reduce atmospheric dinitrogen is limited to prokaryotes. Legumes
and a few other plant species have the ability to fix atmospheric N through
symbiotic relationships. In the case of legumes N2-fixation is carried out by
20 prokaryotes, Rhizobium or Bradyrhizobium in nodules located on the plant
root (Sprent et al., 1990).
Nodulation and the development of an effective symbiosis
is a complex process requiring both bacterial and plant genes. The molecular
mechanisms of recognition between (Brady)rhizobium and legumes can be
25 considered as a form of cell-to-cell interorganismal communication. A precise exchange of molecular signals between the host plant and rhizobia over
space and time is essential to the development of effective root nodules. The
first apparent exchange of signals involves the secretion of phenolic
compounds, flavonoids and isoflavonoids, by host plants (Peters and Verma,
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1990). These signal compounds are often excreted by the portion of the root
with emerging root hairs, a region that is most susceptible to infection by
rhizobia (Verma, ~ 992). These compounds have been shown to activate the
expression of nod genes in rhizobia, stimulating production of the bacterial
5 nod factor (Kondorosi, 1992). This nod factor has been identified as a
lipo-oligosaccharide (Carlson et al., 1993), able to induce many of the early
events in nodule development, including deformation and curling of plant root
hairs, the initiation of cortical cell division, and induction of root nodule
meristems. In soybean for example, the isoflavones, daidzein and genistein,
10 are the major components of soybean root exudates which induce the nod
genes of B. japonicum (Kosslak et al., 1987). Other such substances active
at very low concentrations (10-6 to 10-7 M) have been shown to stimulate
bacterial nod gene expression within minutes. However, the effectiveness of
isoflavonoids is found to vary between cultivars.
Another similarity in the nod region(s) of Rhkobium strains
is the presence of conserved sequence elements within the promoter regions
of certain inducible nod genes. These conserved sequences, first identified
in the nodABC promoter region, are termed the nod-box and are believed to
function in induced nod gene expression, possibly as regulatory protein
20 binding sites.
No Sym plasmids have been associated with
Bradyrhizobium strains. The nitrogenase and nodulation genes of these
bacteria are encoded on the chromosome. Of importance, Bradyrhizobium
strains contain nodulation genes which are reported to functionally
25 complement mutations in Rhizobium and which show significant structural
homology to nodulation gene regions of R. meliloti and R. Ieguminosarum.
The specific components of legume exudate that act to
induce nodulation genes in several species of Rhizobium and Bradyrhkobium
have been identified as flavonoids and related compounds. Luteolin was
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reported to be the component of alfalfa exudates that induces nodABC
expression in R. meliloti. Three clover exudate constituents:
4',7-dihydroxyflavone, geraldone and 4'-hydroxy-7-methoxyflavone were
reported to induce the nodulation genes of R. trifolii. Two pea exudate
5 components: eriodictyol, and apigenin-7-O-glucoside were reported to induce
the nodulation genes of R. Ieguminosarum. In addition, molecules having
structures related to those of the inducer found in exudate were assessed for
their ability to induce. Inducers of Rhizobium nodulation genes appear in
general to be limited to certain substituted flavonoids, and the range of
10 compounds to which a Rhizobium responds is species specific. Since host
range is used to classify Rhizobium strains into different species, this
suggests that differential response to inducer molecules is involved in the
mechanism of determination of host range.
Two isoflavone components of soybean exudate, d~id7ein
15 and genistein, have been reported to be inducers of the nodulation genes of
B. japonicum strains 110 and 123 (Kosslak et al., 1987, Proc. Nathl. Acad.
Sci. USA 84:7482-7432). Several other isoflavones were found to be
inducers (7-hydroxyisoflavone, 5,7-dihydroxyisoflavone and biochanin A) or
weak inducers (formononetin and prunetin) of the B. japonicum nod genes.
20 In addition, two flavones: 4',7-dihydroxyflavone and apigenin which induce
certain Rhizobium nod genes were also found to induce the B. japonicum nod
genes.
In view of the above, it is clear that the exchange of signals
between legume and bacterial strain and intricacies thereof are shared
25 between different legumes and the Rhizobium and Bradyrhizobium genera.
The manner in which nodulation genes are regulated is also conserved
among Rhizobium and Bradyrhizobium strains.
Soybean [Gtycine max (L.) Merr.] is the world's most
widely-produced nitrogen (N) fixing crop. However, soybean is a plant of
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tropical to subtropical origin and, as such, requires temperatures in the 25 to
30~C range for optimal growth and symbiotic N2 fixation. When
well-nodulated, soybean is capable of fixing its own N. Both symbiotic N2
fixation and NO3- utilization appear to be essential for maximum yield. High
soybean yields also require adequate levels of phosphorous and potassium.
Liming acid soils to a pH of 6.0 to 6.5 is an important prerequisite for
profitable soybean production. Adequate populations of Bradyrhizobium
japonicum must be present to produce a well-nodulated soybean crop. Smith
et al. (1981) determined that an inoculum level above 1 x 105 rhizobia per
centimetre of row was necessary to establish effective nodulation.
Root zone temperatures (RZTs) below 25~C strongly and
negatively affect soybean nodulation and N2 fixation (Lynch et al., 1994). In
fact, in short season areas low temperature is considered the major growth
limiting factor for soybean. It has been noted that all stages of nodule
formation and functioning are affected by suboptimal RZTs and experiments
have generally indicated that early nodule development processes are the
most sensitive. The exact mechanism by which suboptimal RZTs affect N
fixation has yet to be identified. Numerous hypothesis have been postulated
however: 1 ) decrease N fixation activity by the nitrogenase enzyme complex;
2) changes in nodule oxygen permeability; 3) rate of export of fixed N from
the nodule; 4) inhibition of N2 fixation inside the nodule; 5) decrease in
bacteroid tissue and/or delay in its rate of formation; 6) via effects on
bacterial physiology and growth; and 7) via effects on plant physiology and
growth.
Production of N fertilizer, in Canada as elsewhere, is
economically ($1 billion per year in Canada), energetically (equivalent to 30
million barrels of oil per year) and environmentally (produce 15 million tones
of CO2 per year, ground water-polluting NO3 and ozone-destroying NOX)
expensive. In eastern Canada the farm community spends approximately
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$150 x 106 per year for N fertilizer. Nitrogen fixation is the sustainable
alternative to N fertilizer. Therefore, an understanding of the mechanism of
suboptimal RZT effects on soybean nodulation and N2 fixation and finding
methods to reduce this restriction by low RZT would allow increased use of
5 this N2-fixing cash crop, and decreased reliance on potentially polluting N
fertilizers in cool season areas. The ability to overcome the negative effects
of suboptimal RZTs could be applied to other stress conditions that
negatively affect nitrogen fixation (water stress, high pH, temperatures etc.).
As well, such an overcoming of stress conditions that negatively affect
10 nitrogen fixation, growth, yield and the like could be adapted to other legumes
and possibly to non-legumes.
Understanding of rhizosphere biology has progressed with
the discovery of a specific group of microorganisms, now called plant growth
promoting rhizobacteria (PGPR), that can colonize plant roots and stimulate
15 plant growth and development (Kloepper et al.,1980a). Most of the identified
strains of rhizobateria occur within gram-negative genera, of which
fluorescent pseudomonads are most characterized, although some strains
of Serratia have been reported (Kloepper et al.,1986,1991; Ordentlich et al.,
1987). Several gram positive strains of root-colonizing bacteria were reported
20 such as an Arthrobacter-like genus (Kloepper et al., 1990) and Bacillus
(Backman et al., 1989; Turner et al., 1991). Other documented PGPR,
include ,4zotobacter species, Azospirillum species, and Acetobacter species
(Brown, 1974, Elmerich,1984; Bashan et al.,1990; Tang,1994).
Several reports related the beneficial effects of PGPR to
25 direct plant growth promotion, disease suppression, associative N2 fixation
and improved access to soil nutrients. Nitrogen fixation promoting
rhizobacteria are identified as PGPR that are capable of increasing
nodulation as well as plant growth. A different approach was taken recently
(Kloepper et al., 1986; Kloepper et al., 1988b) when over 10, 000 bacterial
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strains were randomly isolated from plant rhizospheres at different locations.
After examining their ability to growth at low temperatures, they were tested
under greenhouse conditions, using field soils, for their ability to promote thegrowth and development of soybean and canola plants. Selected strains
5 were then tested in field trials, and some were found to increase growth and
yield. However, some promising strains under lab conditions were not shown
to exhibit their effect in the field. Furthermore, the effect of PGPR strains was
not tested on legumes to assess whether they could somehow increase
nodulation and nitrogen fixation under environmental stress conditions, such
10 as low root zone temperatures (RZT) which retard the onset thereof. It
follows that whether such an effect of PGPRs on nodulation and nitrogen
fixation translated into an increase protein yield and dry matter yield of
legumes grown under such environmental stress conditions was not
determined.
Inoculation of crop seeds with PGPR was of major interest
in agricultural production throughout the twentieth century. The commonly
applied inoculants have involved nitrogen-fixing strains of F~hizobium, which
can form a symbiotic association with legumes and fix nitrogen. The
capability of soil bacteria that are free-living in the soil (non-symbiotic or
"associative") to colonize roots and promote plant growth has been well
documented over the past 30 years (Kloepper et al., 1 988a). Initial efforts to
enhance plant growth with free-living bacteria were centered on either
phosphate-solubilizing bacteria such as Bacillus megaterium var.
phosphoticum or on nitrogen-fixing bacteria that are present in the soil as freeliving bacteria, such as Azotobacter (Cooper, 1959; Brown, 1974). The
free-living phosphate solubilizing and nitrogen-fixing bacteria were shown to
increase the yield up to 25% (Schmidt, 1979). The problem with these
bacteria is their inability to compete effectively with other soil microflora, as
their population density decline after they have been introduced into the
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rhizosphere. This observation established the "biological balance" (Baker et
al., 1974) or"rl,icrobial equilibrium" (Katznelson, 1985) theory, which stated
that the rhizosphere microflora are a distinct collection of organisms that can
live and persist in the soil in equilibrium (Kloepper et al., 1988a). The
assumption was that after new bacteria are introduced into the rhizosphere
their population density will decrease, and the original microbial balance
would be reestablished (Kloepper et al.,1988a).
In the late 1970s, researchers at the University of
California in Berkeley found that the rhizosphere microbiological equilibrium
could be modified by introducing specific strains of rhizosphere
pseudomonads (Kloepper et al.,1988a). These pseudomonads were able to
colonize roots and alter the balance of both fungal and bacterial microflora
throughout the growing season (Kloepper et al., 1980c).
PGPR were reported to increase plant yields 10 to 30% in
non-legume crops such as potato, radish, and sugar beet. Numerous reports
indicated that PGPR can exert precise effects on diverse hosts, including
lentil (Chanway et al., 1989), peanut (Turner et al., 1991), bean (Anderson
et al., 1985), canola (Kloepper et al., 1988b), cotton (Backman et al., 1989;
Greenough et al., 1989, ), pea (Chanway et al., 1989), rice (Sakthivel et al.,
1987), and soybean (Polonenko et al., 1987). The mechanism by which the
PGPR promote plant growth is unknown; however, a wide range of
mechanisms were postulated such as: mobilization of insoluble nutrients (e.g.
phosphate) and resulting enhancement of uptake by the plant (Lifshitz et al.,
1987), associative nitrogen fixation (Chanway et al., 1991a) production of
antibiotics toxic to soil-borne pathogens (De-Ming et al.,1988), production of
plant growth regulators that promote plant growth (Kloepper et al., 1981b;
Gaskins et al., 1985; Neilands et al., 1986 ), siderophore production
[high-affinity iron (Ill) chelator]. Specific Pseudomonad strains have
established yield increases, control of soil-borne plant pathogens, promotion
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of seedling emergence, and promotion of legume nodulation by
nitrogen-fixing (Brady)rhizobium spp under field conditions.
Some reports suggested beneficial effects of some PGPR
on the legume N2-fixing symbiosis; the bacteria involved are known as nodule
5 promoting rhizobacteria (NPR). Inoculation with NPR, often pseudomonads,
and (Brady)rhizobium enhances root nodule number or mass (Singh et al.,
1979; Burns et al., 1981; Grimes et al., 1987; Polonenko et al., 1987;
Yahalom et al.,1987). The ability of NPR to increase nitrogenase activity was
also documented (Iruthayathas et al., 1983; Alagawadi et al., 1988). Burns
10 et al. (1981) reported that co-inoculation of Azotobacter vinlandii and
Rhizobium spp. increased the numbers of nodules on the roots of soybean
(Glycine max), pea (Vigna unguiculata), and clover (Trifolium repens). Both
field and greenhouse data showed that co-inoculation of Pseudomonas
putida increased nodulation of beans (Phaseolus vulgaris) by R. phaseoli
15 (Grimes et al., 1984). The mechanism by which NPR increase nodulation
and/or N2 fixation is obscure and the ecology of NPR is poorly understood.
Different mechanism have been proposed to explain how
rhizobacteria promote plant growth:
(1) by producing plant growth regulators (PGRs), which are organic
20 substances that, at very low concentrations, can influence physiological
mechanisms of the plants. Several soil microorganisms have the capability
to produce active quantities of PGRs, which can effect plant growth and
development. Production of PGRs has been illustrated in both culture media
and in the soil. Plant growth promoting activity by soil microflora can take
25 different forms such as, the production of phytohormones, or fungicidal or
bactericidal indole and phenols. Azospirillum, which is a diazotrophic
bacterium associated with plant roots, particularly with forage grasses and
cereals, in tropical regions, might be of agronomic importance, since it has
repeatedly been reported to promote plant growth (Okon,1994). It is thought
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to benefit plants by the production by phytohormones, e.g. auxins (Hartmann
et al., 1994).
(2) by enhancing phosphate uptake by plants. Considerable research efforts
have been aimed at evaluating phosphate-solubilizing bacteria. Several ways
by which these bacteria, which include fluorescent pseudomonads, may
increase the availability of phosphorus to plants were suggested such as,
mineralization (solubilization of organic phosphate via the action of
phosphatase) or solubilization of unavailable inorganic phosphates by means
of organic acids. Lifshitz et al. (1987) reported that a P. putida PGPR strain
increased the uptake of 32P-labelled phosphate by canola seedlings.
Inoculation of seeds with a pseudomonad PGPR resulted in a significant
increase of 32p levels in roots and in shoots.
(3) By enabling a biological control of soil-borne plant
pathogens. Phytophathogens can reduce crop yields by 25-57%, which is
tremendous loss of crop productivity. Presently, chemical agents (pesticides)
are applied to reduce this loss. Other procedures such as fumigation, steam
treatment, and solarization of soils were also applied (Gamliel et al., 1992).
However, many of these chemicals could have hazardous effects on animals,
including humans, and may persist and accumulate in natural ecosystems.
Currently, biological approaches are being developed to control some plant
pathogens. Such biological approaches include the development of plants
that are able to resist one or more pathogenic agents (Greenberg et al.,
1993) and the use of PGPR that can suppress or prevent the
phytopathogenic damage (O'Sullivan et al., 1992; Sivan et al., 1992; Cook,
1993; Sutton et al., 1993).
The possible use of PGPR as biological control agents is
described by the study of the mode of action for fluorescent pseudomonad
PGPR reported for potato (Kloepper et al., 1981 a). These PGPR strains were
not able to promote plant growth under gnotobiotic conditions (Kloepper et
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al., 1981b). The growth promotion in field soils was accompanied with a
23-64% reduction in the population densities of indigenous rhizoplane fungi
and a 25-93% reduction in gram-positive bacterial population densities
(Kloepper et al., 1981a). Suslow et al. (1982) identified specific strains of
5 root-colonizing bacteria that were pathogenic on sugar beet seedlings and
were termed deleterious rhizobacteria (DRB). Several genera of DRB were
found to cause growth inhibition and root deformations on crop plants
(Suslow et al., 1982; Fredrickson et al., 1985a; Fredrickson et al., 1985b;
Gerhardson et al.,1985; Campbell et al.,1986; Schippers et al., 1987; ).
10Most PGPR strains appear to enhance plant growth
indirectly by reductions in populations of DRB. A study by Kloepper (1983)
demonstrated that inoculation of potato seed pieces with two strains of
fluorescent pseudomonad PGPR, which were responsible for yield increases
in the field, caused a reduction in populations of Erwinia carotovora on roots,
15ranging from 95 to 100% fewer than controls without PGPR treatment. Colyer
et al. (1986) and Xu et al. (1986a,b~ confirmed the biological control of E.
carotovora by selected strains of root-colonizing fluorescent pseudomonads.
A number of such mechanisms by which rhizobacteria
demonstrate biological control have been identified. Generally, bacterial
20 abilities to protect the plants from soil-borne plant pathogens rely on two
aspects: the root-colonization capacity of the biocontrol agent and the
production of siderophores and antibiotics that control the growth of the plant
pathogens. Such mechanisms include:
(1) the production of siderophores. PGPR produce and release siderophore
25 molecules that will bind most of the Fe+3 that is available in the rhizosphere,
and as a result, prevent any pathogens from proliferating because of lack of
iron, and thus facilitate plant growth (O'Sullivan et al., 1992). Pseudomonad
PGPR strains are capable of producing siderophores which chelate the ferric
iron in the rhizosphere thus inhibiting plant pathogenic or deleterious species,
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with less affinity for iron (Kloepper et al., 1980b). An awareness of the
behaviour of introduced PGPR strains and their siderophore-producing
abilities at different temperatures could be of major importance regarding the
use of PGPR for promotion of plant growth. However, the influence of PGPR
strains on plant growth under different climatological conditions needs
additional study before PGPR strains can be introduced into the field.
(2) The production of antibiotics.
(3) Competition among soil microorganisms for infection sites and nutrients.
(4) Hydrolysis of fusaric acid (Toyoda et al.,1991) .
(5) Synthesis of enzymes that are able to hydrolyze the cell walls of fungal
pathogens (Mauch et al., 1988).
It has also been reported that enhancement of plant growth
by rhizobacteria through disease control may comprise direct or indirect
effects on the pathogen (Davison, 1989). Directly by competing with the
pathogen for available nutrients, siderophore production, and production of
antibiotics (Weller, 1988), and indirectly by modifying plant defence
responses. Induced disease resistance is an active resistance mechanism
which depended on the host plant's physical or chemical restrictions,
activated by biotic or abiotic agents (Kloepper et al., 1992).
It is generally understood that root colonization is a
dynamic process and not a temporary relation between bacteria and roots in
soil. It is the process whereby bacteria survive inoculation onto seeds or into
soil, divide and proliferate in response to seed exudates rich in carbohydrates
and amino acids (Kloepper et al.,1985), adhere to the root surface (Suslow,
1982; Weller, 1983), and colonize the root system in soils containing native
microorflora (Kloepper et al., 1980a, Suslow and Schroth, 1982; Weller,
1984). PGPR can multiply and remain in the rhizosphere rc ~ 9 inoculation
onto crop seeds. The bacteria are allocated in the rhizosphere in a log normal
order (Loper et al., 1984) and are sporadically established along the roots
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(Bahme et al.,1987). Colonization, although difficult to measure, is required
for causing an interaction with the plant and other members of the microflora.
The role of root colonization by PGPR has been reviewed (Schroth et al.,
1982; Suslow, 1982).
Rhizosphere colonization symbolizes a larger ecological
niche including root colonization and bacteria that are in close proximity to,
although not necessarily attached to roots (Kloepper et al., 1980a).
Fluorescent pseudomonads, are highly rhizosphere competent (capable of
root colonization), which accounts for their predominance among the PGPR.
Pseudomonads hold several characteristics which assist them in seed
colonization, such as fast growth and motility (Seymour et al.,1973; Arora et
al., 1983; Scher et al., 1985; ). However, these traits may not always
correlate with root colonization. For example, Howie et al. (1987) found that
three nonmotile mutants of P. fluorescens colonized wheat roots as well as
their motile parents.
Rhizosphere colonization has been reviewed recently by
van Elsas et al. (1990), Kloepper et al. (1992) and Kluepfel (1993). The major
problem for successful application of PGPR strains in soil was suggested to
be the lack of constant effectiveness of the inoculant (van Elsas et al.,1990).
Several causes were suggested, such as ineffective colonization of the plant,
or poor survival of the introduced population. Xu et al. (1986b) and Bull et al.(1991), der"ons~l~ted a positive relationship between root colonization by a
PGPR strain and disease suppression, proposing that methodologies which
enhance root colonization may also improve the benefits of a PGPR strains
in soil. The extent and amount of root colonization required by a PGPR strain
in order to enhance plant growth rely on many interrelated considerations.
The choice of methods used to try to increase rhizosphere colonization and
plant growth should take these factors into account (Stephens, 1994b).
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14
As the beneficial bacteria are introduced into the
rhizosphere, they become involved in a web of complex biological
interactions with the host plant and with the surrounding rhizosphere
microorganisms. They obtain their nutrients from the root exudates and are
accordingly dependent on the host plant, while they affect the host by
inducing physiological changes in the plant (Kloepper et al., 1 988b).
Interactions with indigenous rhizosphere microorganisms could take different
forms. It could be neutral, antagonistic (e.g. competition for nutrients,
production of antibiotic compounds, parasitism, or predation) or synergistic
(i.e. the promotion of Rhizobium-induced nodulation of legumes). Several
environmental restrictions, including temperature, moisture, and soil type,
may affect these microbial interactions.
In view of the intrinsic complexity of the PGPR strains
themselves, of the complex relationship with its environment, of the
complexity and unpredictability of environmental factors affecting the
environment, the selection of a particular strain to be tested in the field is at
best an educated guess. Whether a chosen PGPR will survive, colonize the
root and affect growth, nodulation, nitrogen fixation and yield cannot a priori
be predicted.
Nevertheless several such factors can be used in order to
increase the chance of selecting rhizosphere competent strains. Such factors
include: (1) Crop specificity; (2) location on the root; (3) quantity of inoculum
on the seed; (4) co-inoculation of PGPR strains with other microorganisms;
(5) use of carrier materials to improve PGPR survival in the rhizosphere; and
(6) management of the soil.
The ecology of pseudomonad PGPR is a relatively new
research area. Consequently, there is little understanding of how
environmental factors will influence bacterial colonization effects and
persistence on roots and the resulting effects on plant growth. Thus, the
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potential effects of specific PGPR strains on certain legumes, as assessed
by increased nodulation, nitrogen fixation, and yield, based on laboratory
testing can clearly not be directly transposable to the complex field situation.PGPR ability to increase crop yields under diverse field
5 conditions has nevertheless been reported (Schroth et al., 1982; Suslow,
1982; Hemming, 1986; Schippers et al., 1987; de-Freitas et al., 1992).
However, some of these reports demonstrated that seed inoculation with
PGPR does not always lead to yield increases. Possible reasons for this
disagreement, including iron availability, soil-type, and the nature of the soil10 microflora and application procedures have already been discussed.
Other field work has explored the enhancement of seedling
emergence. That specific root-colonizing bacteria can increase seedling
emergence was first reported with strains that caused increases in
emergence rates of soybean and canola seedlings under cold field conditions
15 in Canada (Kloepper et al., 198~). The new class of PGPR strains was
termed emergence-promoting rhizobacteria (EPR). Inoculation of conifer
seeds with Bacillus strains caused increased seedling emergence and
biomass (Chanway et al., 1991b). Chanway (1995) also reported that seed
inoculation with Bacilus polymyxa can result in colonization of western
20 hemlock root systems and increase seedling emergence.
With respect to the promotion of nodulation of legumes, it
was shown that specific pseudomonad strains were able to stimulate
nodulation of leguminous crops by Rhizobium and Bradyrhizobium. Grimes
et al. (1987) reported that a P. pufida strain (M17) increased Rhizobium
25 nodulation of bean in field soils. Similarly, Polonenko et al. (1987) tested the
effects of fluorescent pseudomonads on nodulation of soybean roots by B.
japonicum. However, the effect of low RZT or similar environmental stress
factors were not ~ssessed. In Canadian Patent 1,328,238, Polonenko et al.
(1994) disclose certain PGPR strains which are apparently capable of
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enhancing B. japonic~lm nodulation and soybean plant growth in field soil in
pots, and thus performed under laboratory conditions. These strains were
termed nodulation-promoting rhizobacteria (NPR). Although one field
experiment is reported, there is no teaching or suggestion of the effect of
5 NPR strains in environmental stress conditions such as low RZT; there also
is no tracking or suggestions of the effect of PGPR on nodulation or nitrogen
fixation of legumes under field conditions.
Canadian Patent 1,335,363 (1995) to Kloepper et al.
discloses emergence-promoting rhizobacteria. Such strains, mostly
10 Pseudomonas strains but also Serratia strains (including 1-102) are said to
induce an increase in seedling emergence when soil temperatures were
below 20 degrees centigrade. The results presented in 1,335,363 do not
show increase nodulation and nitrogen fixation, and moreover, they are not
based on field data. Hence, the reported effect of specific PGPR strains on
15 seedling emergence of legumes cannot be transposed to the complex field
situation.
Canadian Patent 1,328,238 (1994) to Kloepper et al.
disclose PGPR strains (mostly Pseudomonas strains but also Serratia strains
including S. proteamaculans strain 1-102 and S. Iiquefaciens strain 2-68) that
20 promote an increase in yield and nodulation under laboratory conditions.
1,328,238 does not teach that the disclosed PGPR act to increase nodulation
and nitrogen fixation under field conditions and/or under stress conditions
such as low RZT.
Due to the number of benefits which can result from the
25 establishment of rhizobia:legume symbiosis, other strategies have been
devised to promote nodulation of legumes.
US patent 4,878,936 to Handelsman et al., teaches a
method for enhancing nodulation of legumes which includes inoculation in the
immediate vicinity of the roots thereof, an effective quantity of bacteria which
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enhance nodulation. However, the results are based on controlled laboratory
conditions, not on field studies. Moreover, the laboratory conditions used,
involved temperatures above 25~C which are not expected to be limiting for
nodulation .
US patent 5,141,745 to Rolfe et al., discloses flavones,
some of which are leguminous plant exudates, which induce expression of
certain nod genes in rhizobium strains. Rolfe et al., however, do not assess
whether their results, all obtained under laboratory conditions, translate into
increase nodulation and growth of the leguminous plant under field
1 0 conditions.
The art is replete with examples demonstrating that results
obtained under the laboratory setting are not predictive of the field situation.Typically, a good controlled environment provides optimal levels of soil
nutrients, soil pH, soil moisture, air humidity, temperature and light. The
plants are usually widely spaced so that they do not compete for light. In
some cases environmental factors such as carbon dioxide may even be
optimized. The field environment is vastly more complicated than that of the
controlled environment setting. The soil will vary in its chemistry and texture
in a fractal pattern, such that, while the soil of a research site can be
cha~acteri~ed in general, it will be variable at every level within the confinesof the experimental area. In a controlled environment setting plants are
usually produced in sterilized rooting media (pasteurized soil, sterile sand, orsome form of artificial rooting media) and there is no soil micro flora or fauna.
Field soil is an ecosystem; it contains an enormous number of bacteria, fungi,
protista, algae, and soil insects. The climate and related atmospheric factors
(light intensity, relative humidity, temperature, rainfall, carbon dioxide
concentration of the air, presence of pollutants) vary constantly under
unpredictable field conditions. Thus, for instance, a researcher may impose
a nutrient li",itdlion in the field, but if the conditions are dry and water is more
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limiting to plant growth than the nutrient in question, there will be no
discernable effect due to nutrient treatments.
The inability to extrapolate from a laboratory to a field
setting is illustrated by work conducted in the 1970's and early 1980's on
5 soybean with strains of Bradyrhizobium japonicum which were hypothesized
to be more energy efficient when fixing nitrogen. Because of the extreme
stability of the triple bond in the dinitrogen molecule nitrogen fixation was
known to be a very energy expensive process (reviewed in Schubert 1982).
In addition, it was discovered that the enzyme which fixed dinitrogen into
10 biologically useful ammonia (nitrogenase) leaked high energy electrons to
protons, so that every time one dinitrogen molecule was fixed into two
ammonia molecules, at least one dihydrogen (the product of two protons plus
two electrons) was produced. This constituted a waste of energy by the
plant-bacterium symbiotic system. Shortly afterward it was discovered that
15 some strains of B. japonicum contained an enzyme that took up the hydrogen
formed and took the high energy electrons back off the protons, hence
recovering much of the energy that would have been lost (Schubert et al.
1978). This lead to speculation that strains containing these "uptake
hydrogenases", referred to as Hup+ strains, would be more efficient and lead
20 to improved plant growth, as the plant would have to supply less energy (as
organic acids) to the bacteria for each ammonia molecule received from
them. Albrecht et al. (1979) compared soybean plants inoculated with Hup+
and Hup- strains of B. japonicum under greenhouse conditions. Average total
nitrogen contents and total dry weights of Hup+ inoculated plants were shown
25 to be larger than those of plants inoculated with Hup- strains. This was
confirmed by Maier et al. (1978). However, under field conditions, Albrecht
et al. (1g79) were unable to detect an increase in dry matter production or
yield between Hup+ and Hup- strains. These results were confirmed by
numerous field condition studies. During the course of these confirmations
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19
however, a superior strain of B. japonicum (532c), which is now included in
almost all soybean inoculants used to produce soybean in Canada, was
identified (Hume et al., 1990). Strikingly, this strain is Hup-.
This example provides a blatant proof involving soybean,
that results obtained in a controlled milieu are a priori not predictive of the
field situation.
PCT patent ~r)p' -~tion WO 94/25568, which was published
November 10,1994 in the name of Rice et al., discloses cold tolerant strains
of Rhizobium which are useful for improving nodulation, nitrogen fixation and
overall crop size under field condltions. However, it is unclear whether the
cold-selected strains indeed provided an advantage to alfalfa, since in certain
experiments the temperate strains performed better than the
cold-temperature selected strain (i.e. Tables 5,6 and 7). This results
corroborates the findings of Lynch et al., 1994 which suggested that
inoculation with B. Japonicum strains from cold environments is unlikely to
enhance soybean N2-fixation under cool soil conditions. Lynch et al., 1994
also suggested that indeed the host plant, and not the bacterial strain,
mediates at least a significant portion of the sensitivity of N2-fixation under
low RZT. Further WO 94/25568 (see below) teaches that commercial
rhizobial inoculants are not consistent in their efficacity and performance, andnodulation failures after use of commercial inoculants are common. This is
explained by the inability of inoculant strains to out-compete indegenous
rhizobial bacteria for root-infection sites, once again demonstrating the
non-predictivity of laboratory results to the field conditions.
US Patent 5,432,079 to Johansen et al., relates to the
isolation of Rhizobium strains having improved symbiotic properties. Once
again this Patent fails to teach an enhancement of growth and or yield of a
legume under field conditions. Moreover, this document is silent on the use
of flavonoids or the like to achieve that goal. It is also silent on the use of
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PGPRs. It teaches however that a higher expression of the nod genes does
not necessarily provide an advantage, but can be detrimental to the
competitive ability of the Rhizobium strains.
It should be noted that to yield maximally a legume which
5 is dependant on nitrogen fixation requires both the correct amount of
nodulation and the correct type of nodules. In general, the best situation for
the plant is to generate a small amount of nodule tissue which is extremely
efficient at nitrogen fixation. If, for some reason, a given crop legume, is
unable to form sufficient nodule tissue, of forms inefficient nodule tissue, its10 yields will be lower than would other wise be the case. During the 1 960's and
early 1970's there was extensive research on the genetics of nitrogen
fixation, from both the bacterial and the plant perspectives. Numerous strains
of (Brady)Rhizobium which formed nodules but fixed little or no nitrogen were
characterized (reviewed extensively in Grant et al., 1973). This could result
in nodule tissue being a larger proportion of the total legume biomass, but
lower legume yield. At a less dramatic level, a study by Lynch et al. (1993)
showed that some strains of Bradyrhizobium produce larger amount of
soybean nodule mass than others, and that some of the "better nodulators"
increased soybean growth, but, some did not. Some strains appeared to
produce more nodule tissue with the same level of nitrogen fixation, while
other strains increased the amount of nodule tissue formed, but this extra
nodule tissue was less efficient than was the case with some other strains
which produced less nodule mass.
Finally, legume plants can produce too much nodule tissue,
even if it is efficient. Carroll et al. (1985) characterized supernodulating
mutants. These mutants form substantially more nodule tissue and fix
substantially more nitrogen than normal legumes, however, this is a sitution
which is out of balance. So much (too much) of the plants energies are
directed toward nitrogen fixation that the growth and yield of these plants is
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less than those normal for the crop types (Carroll et al., 1985; Vest et al.,
1 973).
To date there has been no investigation as to whether
nodulation inhibiting or delaying factors, such as suboptimal RZTs alter the
5 effect of PGPR on legumes under field condition. Further, there has been no
investigation as to a possible compensating role of PGPR on nodulation,
nitrogen fixation and yield under short season conditions.
Elucidation of the mechanisms which affect nodulation and
nodule formation in soybean (or other legumes) under environmental stress
10 factors, such as suboptimal RZTs, and a determination of how to reduce the
negative effects of such stress factors on N2 fixation and symbiosis under
conditions which inhibit or delay this symbiosis would provide a significant
advantage to the production of legumes. For example, it would be
advantageous to understand whether the poor nodulation of soybean at
15 suboptimal RZTs can be compensated for by the use of PGPR.
There thus remains a need to reduce the negative effects
of environmental factors on nodulation and nodule formation and to provide
compositions and methods to enable the enhancement of grain yield and
protein yield of legumes grown under environmental conditions that inhibit or
20 delay nodulation thereof.
The description found herein refers to a number of
documents, the content of which is herein incorporated by reference.
Recent reviews on nodulation factors and Rhizobium
symbiosis are available: Spaink (1995) and Prome et al. (1996).
SUMMARY OF THE INVENTION
The Applicant was the first to demonstrated that PGPR can
compensate for the retardation of nodulation and nitrogen fixation at low
RZTs or under short season conditions. The applicant is also the first to
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demonstrate that PGPR addition can affect early stages in the complex
process leading to nitrogen fixation, under low RZTs.
Before the present invention there had been no
investigations as to whether an inhibitor of the early steps of nodulation, such5 as suboptimal RZTs could be overcome by the addition of PGPR under field
conditions. The Applicant then showed that this technique, applied into the
plant rhizosphere, accelerates the onset of soybean nitrogen fixation and
increases total seasonal fixed nitrogen under field conditions in a short
season area. The results of these experiments further indicated that PGPR
10 addition increases protein and dry matter yield under short season
conditions. Surprisingly, nine PGPR strains which had been selected in cold
environments were tested in the laboratory to verify whether they could
compensate the inhibitory effect of low RZTs, only three strains did. These
three were thus tested in the field and only two demonstrated a potential in
15 increasing nodulation, nitrogen fixation and yield under field conditions.
Thus in a first aspect, the present invention features
compositions comprising PGPR for enhancing nodulation of legumes grown
under environmental conditions which inhibit or delay nodulation thereof. Also
the present invention features compositions comprising PGPR for enhancing
20 protein yield and grain yield of legumes grown under environmental condition
which inhibit or delay nodulation thereof. More particularly, the present
invention features compositions comprising a PGPR strain of the genus
Serratia and even more particularly of compositions comprising a PGPR
strain of Serratia liquefaciens and Serratia proteamaculans. In a particular
25 embodiment, the invention features PGPR strains Serratia liquefaciens 2-68
and Serratia proteamaculans 1-102.
In another aspect, the present invention features
compositions for enhancing nodulation and/or enhancing yield of legumes
grown under environmental conditions which inhibit or delay nodulation
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23
thereof comprising at least one strain of PGPR, and more particularly at least
one strain of Serratia.
In a related aspect, the invention features methods for
enhancing nodulation of legumes grown under environmental condition which
inhibit or delay nodulation thereof, the method comprising an inoculation of
PGPR. Also, the present invention features methods for enhancing protein
yield and grain yield of legumes grown under environmental condition which
inhibit or delay nodulation thereof, the method comprising an inoculation of
PGPR.
In one preferred embodiment, the present invention
features compositions and methods for enhancing protein yield and grain
yield of soybean grown under environmental condition which inhibit or delay
nodulation thereof. In certain embodiments, co-inoculation of soybean with
PGPR and rhizobacteria is disclosed.
In accordance with the present invention, there is provided
a composition for enhancing nodulation of a legume grown under
environmental conditions that inhibit or delay nodulation thereof, the
composition comprising an agriculturally effective amount of a PGPR with a
suitable carrier medium.
In accordance with the present invention, there is also
provided a method for enhancing nodulation of a legume grown under
environmental conditions that inhibit or delay nodulation thereof, comprising:
a) inoculating in the vicinity of one of a seed and root of
said legume with a composition compri~ing an agriculturally effective amount
of a PGPR with a suitable carrier medium.
In accordance with the present invention, there is also
provided a composition for enhancing grain yield and protein yield of a
legume grown under env.l oril)lental conditions that inhibit or delay nodulation
....
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thereof, the composition comprising an agriculturally effective amount of a
PGPR with a suitable carrier medium.
In accordance with the present invention, there is also
provided a method for enhancing grain yield and protein yield of a legume
5 grown under environmental conditions that inhibit or delay nodulation thereof, comprising:
a) inoculating in the vicinity of one of a seed and root of
said legume with a composition comprising an agriculturally effective amount
of a PGPR with a suitable carrier medium.
In accordance with the present invention, there is further
provided a composition for enhancing the rate of emergence of a legume
comprising an agriculturally effective amount of a PGPR strain with a suitable
carrier medium. In a preferred embodiment, the composition for enhancing
the rate of emergence of a legume further comprises a cytokinin such as
1 5 kinetin.
While the instant invention is demonstrated mostly by
experiments performed with Bradyrhizobium japonicum and soybean, the
invention is not so limited. Other legume crops, and rhizobial strains may be
used using the same principle taught herein. Non-limiting examples include,
20 alfalfa, Rhizobium meliloti and a nod gene inducing factor thereof; clover R.meliloti, and a nod gene inducing factor thereof; clover R trifolii, and a nod
gene inducing factor thereof; R. Ieguminosarum peas or lentils, and a nod
gene inducing factor thereof; and beans R. phaesoli and a nod gene inducing
factor thereof. Preferred matching of Rhizobium species with legume crop
groups include:
Rhizobial species Legume crop group
Rhizobium melilotti alfalfa, sweet clover
Rhkobium leguminosarum peas, lentils
Rhizobium phaesolii beans
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Bradyrhizobium japonicum soybeans
Rhizobium trifolii red clover
It will be understood that the composition of the present
invention could contain other stimulating factors such as a number of
flavonoids, isoflavonoids, flavones including flavanones, flavanols and
dihydroflavanols, isoflavones, coumarins kinetin, gibberellic acid and related
molecules.
Nodulation inducing activity was found to reside in a
structurally identifiable group of compounds not limited to those flavones
associated in particular with legumes which include specifically substituted
flavones, flavonones (dihydroflavones), flavanols (3-hydroxyflavones) and
dihydroflavanols. Thus, numerous flavones and dihydroflavanols have
nodulation inducing activity (Hungria et al., 1997; Stacey et al., 1995; Spaink,1995). Although alkoxy substituted flavone other than methoxy have not been
identified from natural sources, there is no reason to believe that alternative
short chain substituents like ethoxy or propoxy groups would abolish
nodulation gene induction activity.
Synthetic as well as natural nodulation gene-inducing
compounds are encorrp~ssed by the scope of the present invention.
Direct or indirect methods of legume inoculation can be
employed. During direct inoculation the bacterium is applied directly to the
seed prior to sowing. This can most simply be accomplished by spraying the
seed with or dipping the seed into a liquid culture containing a desired PGPR
strain.
The concenl~atiGn of the inoculum will be adapted to the
particular situation at hand by the skilled artisan. For example, the skilled
artisan will take into account the level of severity of inhibition or delay of the
environmental conditions on nodulation, the intrinsic activity of a chosen
PGPR, the method of application of the composition, etc.
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26
Hereinafter, the invention is illustrated with reference to B.
japonicum and soybean, but is demonstrative of utility of the invention with
other Rhizobium and Bradyrhizobium species and other legume crop groups.
Indeed, herein the term rhizobia is used loosely and encompasses
5 Rhizobium and Bradyrhizobium species.
The term "PGPR" refers to free living rhizobacteria that, in
the absence of an intimate symbiotic relationship, enhance nodulation,
nitrogen fixation, growth, yield and the like of legumes grown under field
conditions.
The term "environmental conditions which inhibit or delay
nodulation" should be interpreted herein as designating environmental
conditions which postpone or inhibit nodulation and nitrogen fixation and
include, without being limited thereto: conditions that stress the plant, such
as temperature stress, water stress, pH stress as well as inhibitory soil
15 nitrogen concentrations or fixed nitrogen.
As used herein, the recitation "low root zone temperature
(RZT)" as well known to the person of ordinary skill, relates to a temperature
below the optimum temperature of growth, nitrogen fixation (if applicable),
yield and the like of a chosen crop. This optimurn temperature of course
20 varies from crop to crop, and thus so does the low root zone temperature. In
the case of soybean for example which has an optimal temperature for
nodulation and growth between 25~C and 30~C, low RZT is thus any
temperature below about 25~C. It will be understood to the person of ordinary
skill that the above-mentioned conditions which delay nodulation and/or
25 growth of a specific crop will also vary according to the crop and are
conditions which fall outside the optimum range for growth and/or nodulation
of the specific crop. These specific optimum ranges of conditions are well
known in the art. Of course, it will be recognized by the person of ordinary
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skill that a further deviation from the optimum temperature (below 1 7~C in the
case of soybean) can drastically affect growth and nodulation.
As used herein, the term "enhancing protein yield and grain
yield" refers to an enhancement of protein and grain yield of legumes of
5 treated plants in accordance with the present invention or adaptations thereof as compared to control plants.
"An agriculturally effective amount of a composition" for
increasing the growth of legume crops refers to a quantity which is sufficient
to result in a sta~islically significant enhancement of nodulation and/or
10 nitrogen fixation and/or growth and/or of protein yield and/or of grain yield of
a legume crop as compared to the enhancement of nodulation and/or
nitrogen fixation and/or growth, protein yield and grain yield of a control crop.
The term "immediate vicinity of a seed or roots" refers to
any location of a seed or roots wherein if any soluble material or composition
15 is so placed, any exhibit of the plant or of the bacteria, or bacterial cells will
be in actual contact with the seed as it germinates or the roots as they grown
and develop.
The recitation "root associated PGPR" is defined herein as
the ",icrobial population on both the root and the soil attached to the root,
20 while "rhizosphere" is about 5-10 cm region around the plant root where
materials released from the root increase the microbial population and its
activities (Prescott et al., 1993). In accordance with the present invention,
from about 104 to about 10'~ PGPR cells per ml should be inoculated,
preferably between about 106 to about 108 and more preferably about 10
25 cells per ml.
The term "Kinetin" refers to a plant growth regulator which
is a common member of the group of compounds known as cytokinins,
having a core molecule which is adenine-like (Salisbury et al., 1992).
Non-limiting examples of other cytokinins having similar plant growth
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28
regulating activities and encompassed within the scope of the present
invention include zeatin, benzyladenine and isopentyladenine. The
concentration of cytokinin such as kinetin to use in accordance with the
present invention range between about 0.1 ,uM to about 20 I~M, preferably
5 between about 1 ,uM to about 10 ,uM, and more particularly between about
3 I~M to about 8 ,uM.
By ~nodulation gene-inducing" or "nod gene-inducing" is
meant bacterial genes involved in nodule establishment and function.
Representative examples of PGPR strains were deposited
at the ATCC, 10801 University Boulevard, Manassas, Va. PGPR strain of
SerTatia proteamaculans 1-102 and Serratia Liquefaciens 2-68 were granted
ATCC accession numbers and , respectively.
Other objects, advantages and features of the present
invention will become more apparent upon reading of the following non
15 restrictive description of preferred embodiments thereof, given by way of
example only with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Having thus generally described the invention, reference
20 will now be made to the accompanying drawings, showing by way of
illustration a preferred embodiment thereof, and in which:
Figure 1 shows the effects of Kinetin concer,l,dlions on totai
plant dry matter one month after emergence;
Figure 2 shows interaction of Kinetin and PGPR on nodule
25 number at one month after emergence;
Figure 3 shows the effect on Kinetin concentrations on
nodule weight at one month after emergence;
Figure 4 shows the effects of Kinetin concentldt,ons on
nodule number at early pod stage (R3);
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29
Figure 5 shows the interaction of cultivars and the Kinetin
concentration on nodule weight at early pod stage (R3);
Figure 6 shows the interaction of cultivars and Kinetin on
root weight at early pod stage;
Figure 7 shows the effect of cultivars and Kinetin on plant
height at final harvest;
Figure 8 shows the effect of Kinetin on 100-seed weight;
Figure 9 shows the effect of cultivars on seed numbers and
pod numbers;
Figure 10 shows the effect of cultivars on 100 seed weight;
Figure 11 shows the effect of interaction of Kinetin and
PGPR on soybean grain yieid.
Other objects advantages and features of the present
invention will become more apparent upon reading of the following
15 non-restrictive description of preferred embodi",ent~ with reference to the
accompanying drawing which is exemplary and should not be interpreted as
limiting the scope of the present invention.
DESCRIPTION OF THE PREFERRED ~MBODIMENT
In order to test whether PGPR can compensate for the
retardation of nodulation and nitrogen fixation under environmental stress
conditions and to test whether PGPR have an utility in promoting nodulation
nitrogen fixation increasing yield and the like of legumes when grown under
field conditions a number of field tests were carried out.
EXAMPLE 1
Plant ~loutl- promoting rhizobacteria accelerate nodulation and
ificr~ase llilroye.l hxation activity by field grown soybean lGlycine max
(L.) Merr.]
-
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Two field experiments were conducted on two adjacent
sites in 1994 to evaluate the ability of two PGPR strains to increase
nodulation, nitrogen fixation, and total nitrogen yield by two soybean cultivarsunder field conditions in a short season area. The results of these
5 experiments indicated that co-inoculation of soybean with B. japonicum and
PGPR increased soybean nodulation and hastened the onset of nitrogen
fixation during the early soybean growing season, when the soils were still
cool. As a result of the increase in these variables, total fixed nitrogen, fixed
nitrogen as a percentage of total plant nitrogen, and the nitrogen yield also
10 increased due to PGPR application. Inoculation with PGPR only also
increased soybean nodulation and nitrogen fixation by native B. japonicum.
The stimulation of PGPR on legume symbiotic N fixation
and plant growth can be affected by environmental factors, such as RZT.
Under controlled environment conditions, the effect of the PGPR on soybean
15 nodulation and N fixation (see Example 3), and plant growth and
physiological activities (Zhang et al., 1996b) varied with RZT. However, to
date, there have been no investigations of whether co-inoculation of B.
japonicum with PGPR increases soybean nodulation and N fixation under
short season field conditions. Therefore, in this study, we tested the
20 hypotheses that under short season field conditions: 1 ) co-inoculation of B. japonicum with PGPR increases soybean nodulation and N2 fixation when
soybean is planted into cool spring soils, 2) final plant N and protein yield
also increase due to improvement of soybean nodulation and N2 fixation, 3)
inoculation with PGPR only, in the presence of native soil B. japonicum,
25 increases soybean nodulation and nitrogen fixation.
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MATERIALS AND METHODS
Field layout
The two experiments were conducted at the Emile A. Lods
Research Centre, McGill University, Macdonald Campus, Montreal, Canada.
5 Both of the experiments were performed at each of two adjacent sites. One
site was sterilized with methyl bromide (50 9 m~2) under a plastic canopy for
72 h (sterilized site). Three days elapsed between removal of the fumigation
canopy and planting. The other site was kept unsterilized. The sterilized site
was included in this study to prevent possible competition from native B.
10 japonicum or interference from other elements of the soil microflora that
might obscure PGPR effects. The first experiment was designed as a 3 x 2
x 2 factorial organized in a randomized complete block split-plot with four
replications. The main-plot units consisted of PGPR strain applications
(no-PGPR application as a control, Serratia li~uefaciens 2-68 and Serratia
15 proteamaculans 1-102). These two strains were chosen based on the reports
of Zhang et al. (see Example 3 and 1996b). The two soybean cultivars
(Maple Glen and AC Bravor) and the strains of B. japonicum [532C (Hume
et al., 1990) and USDA110] formed the sub-plot units. In the second
experiment, two factors were tested, PGPR application and soybean cultivars
20 with the same design as in experiment 1, except that the two soybean
cultivars were the subplot units. At the sterilized site, each sub-plot was 1.6
x 2 m and consisted of three rows of plants with 40 cm between rows. At the
unsterilized site, each sub-plot (2 x 3 m) consisted of four rows of plants with40 cm between rows. The space between plots was 80 cm and between
25 replications 1 m.
For each replication of both experiments one plot of a
non-nodulating Evans was included for calculation of soil nitrogen availability
and seasonal N2 fixation. The soil type at both sites was a Chicot light sandy
loam. In the previous year, 1993, this experimental field had been planted
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with oat and barley, while in 1992 it was used to produce a crop of green
manure alfalfa. The average nitrogen accumulation in the non-nodulating
control plants was 167 kg ha~'. Potassium and phosphate were provided by
the spring application of 340 kg ha~' of 5-20-20 according to soil test
5 recommendations.
Inoculum preparation
In experiment 1, the inoculum was produced by culturing
B. japonicum strains 532C and USDA110 in yeast extract mannitol broth
(Vincent, 1970) in 2000 mL flasks shaken at 125 rpm at 25~C. The PGPR
strains were cultured in Pseudomonas media (Polonenko et al.1987) in 2000
mL flasks shaken at 250 rpm at room temperature (21-23~C). After reaching
the stable phase (7-days for B. japonicum and 1.5-days for PGPR), both B.
japonicum and PGPR were subcultured. When the subculture reached the
log phase (3-days for B. japonicum and 1-day for PGPR), each of the B.
15 japonicum and PGPR strains was adjusted with distilled water to an A620 (B.
japonicum) and A420 (PGPR) value giving a cell density of 108 cells mL~',
respectively. Equal volumes of B. japonicum and PGPR cultures were mixed
and allowed to stand for approximately half an hour at room temperature
without shaking.
20 Planting methods
Seeds of the soybean cultivars 'Maple Glen' and 'AC
Bravor' were surface-sterilized in sodium hypochlorite (2% solution containing
4 mL L-' Tween 20), then rinsed several times with distilled water
(Bhuvaneswari et al., 1980). These cultivars were selected as they have
25 been developed for production under the short season, cool conditions of
eastern Canada and have performed well there. The seeds were planted by
hand on May 11 and 18 at the unsterilized and sterilized sites, respectively.
The delay in planting the sterilized site was due to the extra time required forthe methyl bromide fumigation. Twenty mL of inoculum (for experiment 1), or
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the same amount of PGPR or distilled water (for experiment 2) per one meter
of row were applied by syringe directly onto the seeds in the furrow. Cross
contamination was prevented throughout planting and all subsequent data
collection procedures by alcohol sterilization of all implements used.
Following emergence seedlings were thinned to achieve a stand of 500,000
plants ha~' (20 plants m~1 of row).
'sN application
To be able to measure the seasonal N2 fixation rates by the
isotope dilution method, 'sN was applied (1.2 kg ha~', 99% pure, Isotec Inc.,
Miamisburg, OH, USA) as double-labelled ammonium nitrate in solution, to
a microplot of six plants (30 x 40 cm) within each subplot in the first three
replications at both the sterilized and unsterilized sites in both experiments.
Each microplot was bordered by plastic sheeting extending 15 cm into the
soil to prevent lateral soil losses of the labelled nitrogen. The labelled
nitrogen was applied at growth stage V1 (the first unifoliate leaf) (Fehr et al.,
1971).
Data collection
One month after planting, the onset of N2 flxation was
tested for. Acetylene reduction activity assays were used as a +/- measure
of nitrogenase activity. From each sub-plot four plants were randomly
selected, uprooted and detopped; the roots then were exposed to 10%
acetylene in a sealed 1 L Mason jar for 10 min. A 0.5 mL gas aliquot was then
extracted and analyzed by gas chromatography (Hardy et al., 1968). As
acetylene reduction activity was detected in all the PGPR application plots,
the number, weight, and nitrogen concentration of nodules were measured.
The final nodulation data were taken from plants harvested
on August 13, when the plants reached reproductive stage 6 [pod(s)
containing a green seed that fills the pod cavity at one of the four uppermost
nodes on the main stem with a fully developed leaf (Fehr et al., 1971)]. The
CA 022~7371 1998-12-04
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34
plants were uprooted, the roots were washed with distilled water and the
nodules were removed, counted and weighed. Microplot materials were
harvested by hand at harvest maturity, oven-dried at 70 jC for at least 48 h,
and weighed. The seeds were threshed by hand and ground using a
Moulinex coffee mill (Moulinex Appliances Inc., Virginia Beach, VA, USA).
The above-ground plant tissue from each microplot was ground to pass a 1
mm screen of a Wiley mill (A. H. Thomas Co., Philadephia, PA, USA). The
nitrogen concer,tlalion of grain and other plant tissues was then determined
by Kjeldahl analysis (Kjeltec system, Tecator AB, Hoganas, Sweden).
Following Kjeldahl analysis, a sample of the di~lillate obtained from the shoot
and grain matèrial of each microplot was dried and the ammonium present
converted to nitrogen gas by the application of the Dumas method (Preston
et al., 1981) before measuring the 'sN-l4N ratio of each sample by emission
spec;l~on,el~y (Jasco N-150 'sN analyzer, Japan Spectroscopic Co., Tokoyo,
Japan). The proportion of the total plant nitrogen derived from N2 fixation was
then determined following the formula described by Lynch et al. (1993):
N% from fixation
= {1 - [('sN:'4N of fixing plant)/('sN:'4N of control plant)]} x 100.
Stali~lical analysis
The data were analyzed stati~lically by analysis of variance
using the Statistical Analysis System (SAS) computer package (SAS Institute
Inc., 1988), except for the onset of nitrogen fixation data (presented in Table
1), which were compared by the Cochran Q test (Hollander et al., 1973).
When analysis of variance showed treatment effects (p~0.05), the least
significant difference (LSD) test was applied to make comparisons among the
means (p~0.05) (Steel et al., 1980).
CA 022~7371 1998-12-04
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RESULTS
Native Soil B. japonicum Levels
At the sterilized site, the fumigation with methyl bromide
was not completely effective and the control plants formed adequate nodules
5 at almost the same rate as the inoculated plants. At the unsterilized site, inexperiment 2, a small number (less than 3 per plant) of large nodules were
formed. Because at least some contamination occurred at both sites, the
non-nodulating plants were used as the reference for esli",ali"g seasonal N2
fixation. Because of the higher levels of contamination at the sterilized site,
10 comparisons between strains could not be made with confidence, therefore,
only the effects of PGPR application, soybean cultivar and the two way
interaction between PGPR application and soybean cultivar were tested.
PGPR effects on nodulation and onset of N2 fixation
Acetylene reduction activity was used as a +/- indicator of
15 the onset of N2 fixation in experiment 1 and showed that inoculation with
PGPR resulted in a two to four days earlier onset of N2 fixation on both sites
(Table 1).
CA 02257371 1998-12-04
W O 98/44802 PCT/CA98100336
36
N ~ _ o S
,C
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CA 022~7371 1998-12-04
W O 98/44802 PCT/CA98/00336
37
B. japonicum USDA110 or 532 C co-inoculated with PGPR
2-68 increased both nodule weight per plant and weight per nodule for both
AC Bravor and Maple Glen at early soybean growth stages relative to
inoculation of the B japonicum strains alone (Table 2). This increase in mass
5 was due to the formation of larger nodules, not to increased nodule number
per plant (Table 2). At the sterilized site, Maple Glen plants receiving PGPR
1-102 caused a 100% increase in the individual nodule weight (Table 3). At
the second sampling, August 13, Maple Glen plants co-inoculated with B.
japonicum USDA110 and PGPR 2-68 had increased nodule numbers and
10 nodule weights per plant compared to the plants receiving B japonicum
USDA110 alone at the unsterilized site (Table 2).
CA 02257371 1998-12-04
W 098/44802 PCT/CA98/00336
38
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CA 022~7371 1998-12-04
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CA 02257371 1998-12-04
W O g8/4~802 PCT/CA98/00336
39
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CA 02257371 1998-12-04
W O 98/44802 PCT/CA98/00336
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CA 022~7371 1998-12-04
W 098/44802 PCT/CA98/00336
Crrecls of PGPR on seasonal Nz fixation
AC Bravor plants co-inoculated with B japonicum
USDA110 and PGPR1-102 increased seasonal N2 fixation by 100% as
compared to plants inoculated with B japonicum USDA110 alone by the N
5 difference method and 92% by the 15N dilution method at the unsterilized site
(Table 4), however, there was no increase in the seasonal N2 fixation for the
same treatment combination at the sterilized site (Table 5). Co-inoculation
with B. japonicum USDA110 and PGPR2-68 increased seasonal N2 fixation
by 100% relative to B. japonicum USDA1 10 alone for AC Bravor plants by the
10 N difference method at both the sterilized and unsterilized sites (Table 4 and
5). The same combination increased the seasonal N2fixation 94% by the 'sN
dilution method at both the unsterilized and sterilized sites (Tables 4 and 5).
Co-inoculation with B. japonicum USDA110 and PGPR1-102 increased the
total plant nitrogen yield of AC Bravor plants by 63% relative to B. japonicum
15 USDA1 10 alone at the unsterilized site (Table 4) but caused no increase at
the sterilized site (Table 5). Co-inoculation with B. japonicum USDA110 and
PGPR2-68 increased total plant nitrogen yield of AC Bravor plants by 50%
relative to B. japonicum USDA110 alone at the unsterilized site (Table 4) and
by 31% increase at the sterilized site (Table 5).
CA 02257371 1998-12-04
WO 98/44802 PCT/CA98100336
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CA 02257371 1998-12-04
W O 98/44802 PCT/CA98/00336
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W O 98/44802 PCTICA98/00336
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CA 022~7371 1998-12-04
W O 98/44802 PCT/CA98/00336
43
Co-inoculation with B. japonicum USDA110 and PGPR
1-102 increased seasonal N2 fixation of Maple Glen plants 30% as estimated
by the '5N dilution method when compared to the B japonicum USDA110
alone at the unsterilized site (Table 4), however, there was no increase in the
seasonal N2 fixation for the same treatment combination at the sterilized site
(Table 5). Maple Glen plants co-inoculated with B. japonicum USDA110 and
PGPR 2-68 increased seasonal N2 fixation levels, estimated by the 'sN
dilution method, 50% higher than those of plants inoculated with USDA110
alone at the unsterilized site (Table 4). The same combination did not
increase the seasonal N2fixation at the sterilized site (Table 5). Interactions
between PGPR application, B. japonicum strains, and soybean cultivars
existed for total fixed N and fixed N as a percentage of total plant N indicating
that 532C and USDA110 had different sensitivities to PGPR preincubation at
the unsterilized site (Table 4). Interactions between PGPR application and
soybean cultivars indicated that AC Bravor tended to be more responsive to
both PGPR treatments for total fixed N, fixed N as a percentage of total plant
N, and N yield in the unsterilized site (Table 5).
PGPR effects on soybean nodulation and N fixation through native B.
japonicum
In experiment 2, where plots were not inoculated with B.
japonicum, application of PGPR onto seeds in the furrow increased the
number of nodules at the sterilized site at the first sampling date (Table 6).
At crop maturity, nodule number was increased by PGPR at the sterilized
site. PGPR application, in the absence of B. japonicum inoculation, also
increased seasonal N2 fixation at both the unsterilized and sterilized sites
(Table 7). The final total nitrogen yield of plants receiving PGPR 2-68 was
57% greater than that if plants receiving only distilled water at the unsterilized
site.
CA 02257371 1998-12-04
WO 98/44802 PCT/CA98/00336
q~ ~ ~ ~ a~ o co c
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WO 98/44802 PCTtCA98/00336
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CA 022~7371 1998-12-04
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46
DISCUSSION
The two PGPR strains S. Iiquefaciens 2-68 and S.
proteamaculans 1-102 used in this study were selected based on work
reported by Zhang et al., (see Example 3 and 1996b), in which nine PGPR
strains were tested for effects on soybean nodulation and nitrogen fixation
over a range of RZTs under controlled environment conditions. In our field
study, the onset of nitrogen fixation by plants receiving bradyrhizobia
preincubated with PGPR was two to three days earlier than those receiving
no PGPR treatment at both the unsterilized and sterilized sites (Table 1),
while PGPR increased the number of nodules per plant at the unsterilized
site. Application of PGPR directly onto the seeds in the furrow at the time of
planting also improved plant nodulation and N2 fixation. The findings of this
field study agreed with results from controlled environment work, in which
co-inoculation of some PGPR with B. japonicum reduced the negative effects
of low RZT on soybean nodulation and nitrogen fixation (Zhang et al., see
Example 3). In addition, a recent work has shown that inoculation of soybean
with PGPRs in the presence and absence of B. japonicum increased
soybean grain yield, grain protein yield, and total plant protein production
under short season areas (unpublished data).
Since nodule dry weight per plant was increased and the
onset of nitrogen fixation was hastened by B. japonicum co-inoculation with
PGPR, total fixed nitrogen and nitrogen yield per plant were increased (Table
5). Sprent (1979) postulated that an increase of ten percent in the period of
nodule activity by a grain legumes, particularly between the onset of N2
fixation and the attainment of maximum fixation, could double the seasonal
level of nitrogen fixed. In our experiment, the period of nodule function was
about 70 days (late-June to early-September). PGPR application resulted in
a two to four day increase in the duration of N2 fixation (Table 1). PGPR
application increased the total amount of nitrogen fixed. It seems likely that
CA 022~7371 1998-12-04
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some of this increase in total fixed nitrogen was due to earlier nitrogen
fixation, hastened by PGPR application under short season conditions where
soils were stressfully cool in the early growing season, with the remainder
being due to increased nodules mass.
The mechanisms of growth and nitrogen fixation promotion
by PGPR are not well understood; however, a wide range of possibilities
have been postulated, including both direct and indirect actions. Direct
actions include an increase in mobilization of insoluble nutrients and
subsequent enhancement of uptake by the plants (Lifshitz et al., 1987),
production of antibiotics toxic to soil-born pathogens (Li et al., 1988), and
production of plant growth regulators that stimulate plant growth (Gaskins et
al., 1985). Indirect actions include positive effects on symbiotic nitrogen
fixation through enhanced root n~dule number or mass (Grimes et al., 1984;
Yahalom et al., 1987; Zhang et al., see Example 3) and increased
1S nitrogenase activity (Iruthayathas et al., 1983; Alagawadi et al., 1988).
The results of experiment 2, which investigated the effects
of PGPR application without B. japonicum addition on soybean growth and
development, were different from those observed in experiment 1. At the
unsterilized site, although both PGPRs did not increase plant nodule number,
they both increased weight per nodule, total fixed nitrogen and N yield
(Tables 6 and 7). At the sterilized site, both S. Iiquefaciens 2-68 and S.
proteamaculans 1-102 increased nodule number, total nitrogen fixed, and N
yield.
The average proportional increases in nodule number per
plant, nodule weight per plant, total fixed nitrogen, and N yield were generallylarger in experiment 1 than in experiment 2. There are two possible
explanations for this observation. First, in experiment 1, PGPR were
perincubated with B. japonicum for a period of at least 30 minutes before
inoculation, during which PGPR-B. japonicum interactions might have
CA 022~7371 1998-12-04
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48
occurred. Plant growth promoting rhizobacteria produce many
phytohormones and signal molecules (Burr et al., 1984; Davison, 1988;
Kapulnik, 1991), such as genistein, the plant-to-bacteria signal involved in thesoybean nodule infection and formation processes. Therefore, inoculation of
5 soybean plants with B. japonicum and PGPR could have resulted in higher
relative increases in nitrogen fixation and subsequent soybean growth and
yield than B. japonicum or PGPR alone. Second, the PGPR may have
stimulated overall plant growth, leading to greater nitrogen demand by the
developing soybean plants, leading in turn to greater nodulation and nitrogen
10 fixation. The data of Zhang et al. (1996b), showing improved soybean
photosynthesis and growth due to PGPR prior to the onset of nitrogen
fixation, argue against the former of these two possibilities.
The cultivar AC Bravor tended to be more responsive to
inoculation with PGPR plus B. japonicum than Maple Glen at the sterilized
15 site (Tables 3 and 5). AC Bravor is a later-maturing cultivar and has a higher
potential yield than Maple Glen (Conseil Des Productions Végétales du
Québec recommendations); however, at crop physiological maturity it had
lower nodule numbers, nodule weight per plant and total nitrogen yield than
Maple Glen at the sterilized site (Tables 3 and 5). Therefore, there was less
20 likely to be a nitrogen li"lilation to the growth and development of Maple Glen
than AC Bravor. The increased nodule number and dry matter per plant of
AC Bravor due to PGPR application would have reduced any nitrogen
limitation; therefore, the increases in total fixed nitrogen and nitrogen yield
were greater than those of Maple Glen. At the unsterilized site, the same
25 pattern was found for nodule number and nodule weight per plant for PGPR
1-102 (Table 2). PGPR 2-68 showed a different pattern as it increased
nodule number and nodule weight per plant for Maple Glen plants. An
interaction also existed between PGPR strain and B. japonicum strain
(Tables 2 and 4). The combination of USDA110 with PGPR 2-68 or 1-102
CA 022~7371 1998-12-04
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49
resulted in greater increases in total fixed nitrogen, fixed nitrogen as a
percentage of total plant nitrogen, and nitrogen yield than was the case for
strain 532C (Table 4).
In the second experiment, plots were not inoculated with
5 B. japonicum, and inoculation with PGPR did not increase nodule number at
the unsterilized site. However, nodule number increased in the early growing
season and at the final harvest at the sterilized site (Table 6). This resulted
in an increase in the total fixed nitrogen, tissue N concentration, and N yield
(Table 7). It seems that the PGPRs were able to stimulate the native soil B.
10 japonicum, resulting in increased soybean nodulation and N2 fixation in the
absence of other soil microflora which might have interfered with this effect.
In summary, this is the first field experiment showing that
co-inoculation of B. japonicum with PGPR increased soybean nodulation and
nitrogen fixation. PGPR application increased nodule dry matter per plant and
~5 hastened the onset of nitrogen fixation, especially for early-planted soybean(unsterilized site). Total fixed nitrogen, fixed nitrogen as a percentage of total
plant nitrogen, and total nitrogen yield were all increased in at least some
cases due to PGPR application. Interactions existed between PGPR
application and soybean cultivar indicating that PGPR applied to potentially
20 more nitrogen-stressed plants was more effective. Overall, from this study it was clear that preincubation of B. iaponicum with PGPR can increase
soybean nodulation and nitrogen fixation in the early part of the growing
season when soil temperatures are low.
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EXAMPLE 2
Application of plant growth-promoting rhizobacteria to soybean
(Glycine max [L.] Merr.) increases protein and dry matter yield under
short season conditions
We previously reported that application of plant
growth-promoting rhizobacteria (PGPR) increased soybean growth and
development and, specifically, increased nodulation and nitrogen fixation over
a range of root zone temperatures (RZTs) in controlled environment studies.
In order to expand on the previous studies, field experiments were conducted
on two adjacent sites, one fumigated with methyl bromide and one
nonfumigated, in 1994. Two experiments were conducted at each site, one
involving combinations of two soybean cultivars and two PGPR strains, the
other involving the same factors, but also in combination with two strains
Bradyrhizobium japonicum. Soybean grain yield and protein yield were
measured. The results of these experiments indicated that co-inoculation of
soybean with B. japonicum and Serratia liquefaciens 2-68 or Serratia
proteamaculans 1-102 increased soybean grain yield, protein yield, and total
plant protein production, compared to the nontreated controls, in an area with
low spring soil temperatures. Interactions existed between PGPR application
and soybean cultivar, suygesti"g that PGPRs applied to cultivars with higher
yield potentials were more effective. PGPRs applied into the rhizosphere
without addition of B. japonicum also increased plant grain yield 22%, protein
yield 13% and total plant protein 23%. Overall, inoculation of soybean plants
with PGPRs in the presence and absence of B. japonicum increased
soybean grain yield, grain protein yield, and total plant protein production
under short season conditions.
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MATERIALS AND METHODS
Field layout and site preparation
Two experiments were conducted at the Emile A. Lods
Research Centre, McGill University, Macdonald Campus, Montreal, Canada.
5 They were performed at two adjacent sites. One site was fumigated with
methyl bromide (50 g m~2) applied under a plastic canopy for 72 hours, to
prevent possible interference from soil microfloral or faunal elements that
might obscure PGPR treatment effects. Three days elapsed between
removal of the fumigation canopy and planting. The soil of the other site was
10 left nonfumigated.
The experimental design was a 3 x 2 x 2 factorial organized
in a randomized complete block split-plot with four replications. The first
experiment included three factors, PGPR application, B. japonicum strain,
and soybean cultivar. The main-plot units consisted of PGPR strain
15 applications (no-PGPR application as a control, Serratia liquefaciens 2-68
and Serratia proteamaculans 1-102). The two strains tested were chosen
based on the results of a previous controlled environment experiment (Zhang
et al., 1996c). The subplot units were formed by the combination of soybean
cultivars and B. japonicum strains. The soybean cultivars, Maple Glen and
20 AC Bravor were selected as they have been developed for production under
the short season, cool conditions of eastern Canada. Both are widely grown
and yield well in this area. The B. japonicum strains tested were 532C (Hume
et al., 1990) and USDA110, both of which are or have been included in
commercial inoculants used in eastern Canada. In the second experiment
25 two factors were tested, PGPR strains, and soybean cultivars. The design of
the experiment was the same as experiment 1. Three levels of PGPR
applicalions (no-PGPR control, S. Iiquefaciens 2-68, and S. proteamaculans
1-102) formed the main plot units, and two soybean cultivars (Maple Glen
and AC Bravor) were the subplot units. At the nonfumigated site, each
,
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52
sub-plot (2 x 3 m) consisted of four rows of plants with 40 cm between rows.
The space between plots was 80 cm and between replications 1 m. At the
fumigated site, each sub-plot was 1.6 x 2 m and consisted of three rows of
plants, also with 40 cm between rows. The space between plots was 80 cm
5 and between replications, 2 m. The soil type at both sites was a Chicot light
sandy loam. In the previous year, 1993, this experimental field was planted
with oat and barley, while in 1992 it was used to produce a crop of green
manure alfalfa. The soil nitrogen available for soybean uptake proved to be
reasonably high. The average nitrogen accumulation in non-nodulating
10 soybean plants seeded at the same site was 167 kg ha~'. Potassium and
phosphate were provided by spring application of 340 kg ha 1 of 5-20-20, N,
P2O5, K2O, according to a soil test.
Inoculum preparation
For experiment 1, the inoculum was produced by culturing
B. japonicum strains 532C and USDA110 in yeast extract mannitol broth
(Vincent, 1970) in 2000 mL flasks shaken at 125 rpm at room temperature
(23-25~C). The PGPR strains were cultured in Pseudomonas media
(Polonenko et al. 1987) in 2000 mL flasks shaken at 250 rpm at room
temperature (21-23~C). After both B. japonicum and PGPR reached the
20 stationary phase (7-days for B. japonicum and 1.5-days for PGPR), they
were subcultured under the same conditions as described above. When the
subculture reached the log phase (3-days for B. japonicum and 1-day for
PGPR),B. japonicum and PGPR strains were each adjusted with distilled
water to an A620 and A420 value, respectively, giving a cell density of 2 x 108
25 cells mL~'. Before inoculation equal volumes of B. japonicum and PGPR
cultures were mixed and allowed to stand for a minimum of 30 min at room
temperature.
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Planting methods
Seed of the soybean cultivars 'Maple Glen' and 'AC Bravor'
were surface-sterilized in sodium hypochlorite (2% solution containing 4 mL
L-' Tween 20), then rinsed several times with distilled water (Bhuvaneswari
et al., 1980). The seeds were planted by hand on May 11 and 18 at the
nonfumigated and fumigated sites, respectively. The delay in planting the
fumigated site was due to the extra time required for the methyl bromide
application. Twenty mL of combined B. japonicum-PGPR inoculum (for
experiment 1), or the same volume and cell density of PGPR inoculum or the
same volume of distilled water (for experiment 2) per one meter row were
applied by syringe directly onto the seed in the furrow. Cross contamination
was prevented throughout planting and all subsequent data collection
procedures by alcohol sterilization of all implements used. Following
emergence seedlings were thinned to achieve a stand of 500,000 plants ha~'
(20 plants m~' of row).
Data collection
Plant samples were taken on August 13, at which time
plants were at reproductive stage 6 (pod containing a green seed that fills the
pod cavity at one of the four uppermost nodes on the main stem with a fully
developed leaf [Fehr et al., 1971]), to investigate growth variables, such as
leaf number, leaf area, pod number and seed number. Leaf number and area
per plant were determined using a Delta-T area meter (Delta-T Devices Ltd.,
Cambridge, UK). Pod number and seed number per plant were counted by
hand. Grain yield was determined from a one meter row of plants taken from
the middle row of each plot. Plants were harvested by hand at harvest
maturity, then shelled with a plot combine (Wintersteiger, Salt Lake City, UT),
oven-dried at 70~C for at least 48 hours, and weighed. Six additional plants,
also from the middle row, were hand-harvested, and oven-dried at 70~C,
after which seeds were manually separated from shoots. Total shoot weight
CA 022~737l l998-l2-04
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54
and harvest index were determined from these plants, which had been
enclosed within wire mesh from flowering to maturity to allow the collection
of senescent leaves. The dried seeds from each plot were ground using a
Moulinex coffee mill (Moulinex Appliances Inc., Virginia Beach, VA, USA).
5 The nitrogen concentration of seeds was then determined by Kjeldahl
analysis (Kjeltec system, Tecator AB, Hoganas, Sweden). The protein
concentration was calculated by multiplying the nitrogen concentration by
6.25.
Statistical analysis
Results were analyzed statistically by analysis of variance
using the Statistical Analysis System (SAS) computer package (SAS Institute
Inc., 1983). When analysis of variance showed significant treatment effects,
the least significant difference (LSD) test was applied to make comparisons
among the means at the 0.05 level of significance (Steel et al., 1980).
RESULTS
Temperature and seed emergence
The average daily temperature for both air and soil (at a
depth of 5 cm) was below 15~C through early June, and remained below
20~C until mid-July. These conditions slowed the rate of seedling emergencel
particularly for the plants at the nonfumigated site (early planted). For the
May 1 1 planting, at the nonfumigated site, seedlings emerged on May 25,14
days after planting. At the fumigated site, the seeds were planted on May 18
and the seeding emerged at 9 DAP, only 2 days after the nonfumigated site.
25 Experiment 1
The nodule number of uninoculated plants in experiment
2 indicated that the native soil population of B. japonicum in the nonfumigated
soil was low, with uninoculated plants forming few nodules. However, at the
fumigated site, fumigation with methyl bromide was not completely effective
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and the un-inoculated plants in experiment 2 formed nearly as many nodules
as the inoculated plants in experiment 1. Therefore neither of the two
interactions relating to B. japonicum strain nor the three way interaction were
tested at the fumigated site.
Many growth variables, such as plant height, time of crop
maturity, harvest index, and seed moisture content at harvest maturity, were
not affected by the inoculation of PGPR at either site (data not shown).
Overall, leaf number was increased by PGPR application. The leaf area of
AC Bravor receiving USDA110 was increased by inoculation with a mixture
of B. japonicum and PGPR at the nonfumigated site (Table 8), while at the
fumigated site leaf area was increased by PGPR application across all levels
of B japonicum strain and soybean cultivar, except for PGPR 2-68 with
Maple Glen (Table 9). Differences between the two PGPR strains were not
observed for any plant growth variables at either site.
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56
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CA 022~7371 1998-12-04
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CA 02257371 1998-12-04
WO 98/44802 PCT/CA98/00336
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58
Averaged over the two PGPR strains, the number of seeds
formed on AC Bravor inoculated with a mixture of B. japonicum USDA110
and PGPR increased by 52% at the nonfumigated site (Table 8), while at the
fumigated site the number of seeds of AC Bravor plants receiving B.
5 japonicum and PGPR increased by 58% (Table 9). This increase in seed
number was due to increases in their pod numbers (Tables 8 and 9). Since
seed number per plant increased and seed protein concentration did not
decrease, total grain yield and protein yield also increased. At the
nonfumigated site, averaged over the two PGPR strains, the final grain yield
10 of AC Bravor and Maple Glen inoculated with a mixture of B. japonicum
USDA110 and PGPR increased by 23 and 21%, respectively (Table 8). At
the fumigated site the grain yields of AC Bravor receiving B. japonicum and
either S. proteamaculans 1-102 or S. Iiquefaciens 2-68 were 23, and 29%
higher than plants receiving only B. japonicum (non-PGPR control plants),
15 respectively (Table 9). Again, there was no difference between the two PGPR
strains for increase in soybean grain yield.
The main effect of PGPR on protein concentration was
significant at both sites. Averaged over all the treatments, the protein
concentrations of plants receiving PGPR application were 7.5, and 5.1%
20 higher than those of the non-PGPR control plants at the nonfumigated and
fumigated sites, respectively (Tables 8 and 9). Because both final grain yield
and protein concentration were increased by inoculating with a mixture of B.
japonicum and PGPR, the final grain protein and total plant protein yields
also increased at both sites. At the nonfumigated site, the grain protein yield
25 of AC Bravor receiving the mixture of USDA110 and S. proteamaculans
1-102 was 22% higher than its corresponding control plants (Table 8). At the
fulll ;,?t~d site, the protein yield of AC Bravor plants receiving S. Iiquefaciens
2-68, and S. proteamaculans 1-102 application increased by 60, and 50%
over AC Bravor receiving only B. japonicum (Table 9). The total plant protein
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59
yield increase due to PGPR application generally followed the same pattern
as the grain protein yield (Tables 8 and 9).
Two way interactions between either PGPR strain and
soybean cultivar, or PGPR strain and B. japonicum strain existed for most of
5 the yield related variables investigated in these studies at both sites (Tables
8 and 9). The combination of AC Bravor and B. japonicum USDA110 was
more sensitive to PGPR application than Maple Glen and 532C.
Experiment 2
Plant growth-promoting rhizobacteria, directly applied onto
10 seeds in the furrow at the time of planting, did not have any effect on growth
variables and yield compared to control plants at the nonfumigated site
(Table 10) but increased these variables at the fumigated site (Table 11).
Generally speaking, at the fumigated site, the effects of PGPR application
directly into rhizosphere soil, on soybean growth variables, yield components,
15 and final grain and protein yield followed the same pattern observed in
experiment 1. The combination of PGPR 2-68 increased leaf area (36%),
seed number (32%), and grain protein yield (20%) of AC Bravor piants. The
increase in the grain protein yield was not due to any increase of grain
protein concentration.
CA 02257371 1998-12-04
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CA 022~737l l998-l2-04
WO 98/44802 PCT/CA98/00336
61
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62
DISCUSSION
Plant growth-promoting rhizobacteria strains S. Iiquefaciens
2-68 and S. proteamaculans 1-102 were selected following previous
controlled environment studies (Zhang et al., see Example 3) in which nine
PGPR strains were tested for effects on soybean plant growth, development,
nodulation and nitrogen fixation over a range of RZT under controlled
environment conditions. Specifically, S. Iiquefaciens 2-68 performed well at
optimal RZT (25~C), while S. proteamaculans 1-102 performed best at
suboptimal RZTs ranging from 18 to 15~C. In our current studies,
10 co-inoculation with PGPR and B. japonicum improved plant growth,
development, yield components, and final grain and protein yield in the
presence and absence of methyl bromide fumigation. Application of PGPR
with the B. japonicum directly onto the seeds in the furrow at the time of
planting also improved plant growth and increased grain and protein yield at
the fumigated site. These results agreed with those previously found under
controlled environment conditions (Zhang et al., see Example 3). However,
the effects of PGPR application on plant growth, development, and final grain
and protein yield were not different between S. Iiquefaciens 2-68 and S.
proteamaculans 1-102. This could be due to variations in field soil
temperature during the entire soybean growing season.
Inoculation of soybean plants with a mixture of B.
japonicum and PGPR not only increased plant dry matter accumulation, but
also increased grain protein and total protein production at both sites in
experiment 1 (Tables 8 and 9) and at the fumigated site in experiment 2
(Table 11). Zhang et al. (see Example 3) reported that co-inoculation of some
PGPP< with B. japoniucm could reduce the negative effects of low RZT on
soybean nodulation and nitrogen fixation. In addition, a recent study at McGill
University found that co-inoculation of PGPR and B. japonicum accelerated
the processes of soybean nodulation and the onset of nitrogen fixation under
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63
short season field conditions. Sprent (1979) postulated that an increase of
10% in the period of nodule activity of a grain legume, particularly between
the onset of nitrogen fixation and the attainment of maximum fixation, could
double the seasonal level of nitrogen fixation. In a controlled environment
5 experiment, the onset of nitrogen fixation by plants co-inoculated with B.
japonicum and the most effective PGPR strains began 2 to 3 days earlier
than those receiving only B. japonicum (Zhang et al., see Example 3).
Therefore, it is possible that application of PGPR increased grain and total
protein yield under field conditions.
The mechanisms of growth and nitrogen fixation promotion
by PGPR are not well understood; however, a wide range of possibilities
have been postulated, including both direct and indirect actions. The direct
actions include an increase in mobilization of insoluble nutrients and
subsequent enhancement of uptake by the plants (Lifshitz et al., 1987),
production of antibiotics toxic to soil-born pathogens (Li et al., 1987), and
production of plant growth regulators that stimulate plant growth (Gaskins et
al., 1985). Indirect actions include positive effects on symbiotic nitrogen
fixation by enhanced root nodule number or mass (Grimes et al., 1984;
Yahalom et al., 1987; Zhang et al., see Example 3) and increased
nitrogenase activity (Iruthayathas et al.,1983; Alagawadi et al., 1988).
The results of experiment 2, which investig~ted the effects
of PGPR application without B. japonicum addition on soybean growth and
development, were different from those observed in experiment 1. At the
nonfumigated site, although both PGPR S. Iiquefaciens 2-68 and S.
proteamaculans 1 -102 numerically increased plant growth variables such as
leaf area and seed numbers, there were no stalislically significant differences
among treatments (Table 10). At the fumigated site, both S. Iiquefaciens 2-68
and S. proteamaculans 1 -102 affected most plant variables, especially on AC
Bravor plants. As described above, the native soil population of B. japonicum
CA 022~7371 1998-12-04
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64
in nonfumigated soil was low, with uninoculated plants forming few nodules,
whereas at the fumigated site, uninoculated plants formed nearly as many
nodules as the inoculated plants in experiment 1. Therefore, the difference
in results between the sites may indicate that the PGPR were not able to
5 perform well in the absence of B. japoniucm under short season field
conditions.
The average proportional increases in plant growth
variables, yield components and final grain and protein yield were generally
larger in experiment 1 than in experiment 2. There are two possible
10 explanations for this observation. First, as described above, PGPR
application may not have acted effectively in the absence of B. japonicum
under short season field conditions. Second, in experiment 1, PGPR were
preincubated with B. japonicum for a period of at least 30 minutes before
inoculation, during which a number of PGPR-B. japonicum interactions might
15 have occurred. Plant growth promoting rhizobacteria produce many
phytohormones and signal molecules (Burr et al., 1984; Davison, 1988;
Kapulnik, 1991), such as genistein, a plant-to-bacteria signal involved in the
soybean nodule infection and formation processes. Preincubation of B.
japonicum inocula with genistein increased nodule number and hastened the
20 onset of nitrogen fixation at suboptimal RZT under both controlled
environment (Zhang et al., 1995c) and short season field (Zhang et al.,
19g6b) conditions. Therefore, co-inoculation of soybean plants with B.
japonicum and PGPR could have resulted in higher relative increases in
nitrogen fixation and subsequent soybean growth and yield than with B.
25 japonicum or PGPR alone.
.,
CA 022~7371 1998-12-04
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EXAMPLE 3
Growth, Survival, and Root Colonization of Plant Growth-Promotin~
Rhizobacteria under short season conditions
Co-inoculation of B. japonicum with plant growth promoting
5 rhizobacteria (PGPR) has been shown to increase soybean [Glycine max (L.)
Merr.3 nodulation, nitrogen fixation, growth, and development compared to
controls not inoculated with PGPR, in an area with low spring soil
temperatures. We studied the growth and survival of rifampacin resistant
strains of two plant growth promoting rhizobacteria (PGPR) Serratia
tiquefaciens 2-68 and Serrafia proteamaculans 1-102 inoculated on soybean
plants under cool spring conditions. Two field experiments were conducted
on two adjacent sites, one fumigated with methyl bromide and one not
fumigated, in 1994. Two experiments were conducted at each site, one
involving combinations of two soybean cultivars, two strains of
15 Bradyrhizobium japonicum and two PGPR strains, the other involving the
same factors, but without B. japonicum. The population density of PGPR
applied into the rhizosphere without addition of B. japonicum increased over
time indicating that the PGPR were able to survive and proliferate. Overali,
PGPR inoculated onto soybean roots was able to grow, survive, and colonize
20 the roots better at the fumigated site. PGPR 2-68 achieved higher population
densities on both the root and in the soil (rhizosphere) which demonstrates
their ability to colonize the root more rapidly.
MATERIALS AND METHODS
25 Field layout and site preparation
Two experiments at two adjacent sites were conducted at
the Emile A. Lods Research Centre, McGill University, Macdonald Campus,
Montreal, Canada. One site was fumigated with methyl bromide (50 9 m~2)
applied under a plastic canopy for 72 hours, to prevent possible interference
CA 02257371 1998-12-04
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66
from soil microfloral or faunal elements. Three days elapsed between
removal of the fumigation canopy and planting. The soil of the other site was
left nonfumigated.
The experiment was arranged in a 3 x 2 x 2 factorial
5 organized in a randomized complete block split-plot with four replications.
Experiment 1 included three factors, PGPR application, B. japonicum strain,
and soybean cultivar. The main-plot units consisted of PGPR strain
applications (Serratia liquefaciens 2-68 and Serratia proteamaculans 1-102).
The two strains tested were chosen based on the results of a previous
10 controlled environment experiment (Zhang et al., 1 996b). The subplot units
were formed by the combination of soybean cultivars and B. japonicum
strains. The soybean cultivars, Maple Glen and AC Bravor were selected as
they have been developed for production under the short season, cool
conditions of eastern Canada. Both are widely grown and yield well in this
area. The B. japonicum strains tested were 532C (Hume and Shelp, 1990)
and USDA110, both of which are or have been included in commercial
inoculants used in eastern Canada. In experiment 2, only two factors were
tested, PGPR strains, and soybean cultivars. The design of experiment 2
was the same as experiment 1. Two levels of PGPR (S. Iiquefaciens 2-68,
and S. proteamaculans 1-102) formed the main plot units, and two soybean
cultivars (Maple Glen and AC Bravor) were the subplot units.
Inoculum preparation
For experiment 1, the inoculum was produced by culturing
B. japonicum strains 532C and USDA110 in yeast extract mannitol broth
(Vincent, 1970) in 2000 mL flasks shaken at 125 rpm at room temperature
(23-25~C). Two rifampacin resistant PGPR strains t1~102 Serratia
proteamaculans and 2-68 Serratia liquefaciens) were tested in this
experiment. The PGPR strains were cultured in Pseudomonas media
(Polonenko et al. 1987) in 2000 mL flasks shaken at 250 rpm at room
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temperature (21-23~C). After both B. japonicum and PGPR reached the
stationary phase (7-days for B. japonicum and 1.5-days for PGPR), they
were subcultured under the same conditions as described above. When the
subculture reached the log phase (3-days for B. japonicum and 1-day for
5 PGPR), B. japonicum and PGPR strains were each adjusted with distilled
water to an A620 and A420 value, respectively, giving a cell density of 2 x 1 o8cells mL~'. Before inoculation equal volumes of B. japonicum and PGPR
cultures were mixed and allowed to stand for a minimum of 30 min at room
temperature.
10 Planting methods
Seed of the soybean cultivars 'Maple Glen' and 'AC Bravor'
were surface-sterilized in sodium hypochlorite (2% solution containing 4 mL
L-1 Tween 20), then rinsed several times with distilled water (Bhuvaneswari
et al., 1980). The seeds were planted by hand on May 11 and 18 at the
15 nonfumigated and fumigated sites, respectively. The delay in planting the
fumigated site was due to the extra time required for the methyl bromide
application. Twenty mL of combined B. japonicum-PGPR inoculum (for
experiment 1), or the same volume and cell density of PGPR inoculum (for
the treatment) or the same volume of distilled water (for the control) (for
20 experiment 2) were applied by syringe directly onto the seed in the furrow.
Cross contamination was prevented throughout planting and all subsequent
data collection procedures by alcohol sterilization of all implements used.
Enumeration of PGPR
Root and soil samples were collected twice during the
25 experiment. The first sample was collected June 20 when the plants were
beginning to bloom [one open flower at any node on the main stem(R1)]. The
second sample was collected on August 13 when the plants had reached
reproductive stage 6 [pod(s) containing a green seed that fills the pod cavity
at one of the four uppermost nodes on the main stem with a fully developed
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68
leaf] (Fehr et al., 1971). Number of culturable PGPR cells in the bulk soil
(rhizosphere) was determined by transferring 10 g of the soil into a 250-mL
Erlenmeyer flask, containing 90 mL of sterile distilled water. The flasks were
shaken for 30 min at room temperature (200 rpm). Serial 10-fold dilutions
were made and plated on King's B agar (proteose peptone, 20 g; K2HPO4,
1.5 g; MgSO4 7H2O,1.5 9; glycerol,10 9; agar,15 9; deminerilized water,1L;
pH 7.2) supplemented with 100 mg L-' cycloheximide (to inhibit fungal
growth) and 100 mg L~' rifampacin. Plant roots and adhering soil were
seperated from the bulk soil (rhizosphere) by careful, manual shaking. Roots
and adhering soil were then transferred into a 250-mL Erlenmeyer flask,
containing 90 mL of sterile distilled water. Shaking, dilution and plating
procedures were similar to those desribed for the bulk soil (rhizosphere). The
plates were incubated for 24 hr and the cfu g-1 of dry soil and root were
calculated.
Sta~istical analysis
Results were analyzed s~atislically by analysis of variance
using the Statistical Analysis System (SAS) computer package (SAS Institute
Inc., 1983). When analysis of variance showed significant treatment effects,
the least si~nificant difference (LSD) test was applied to make comparisons
among the means at the 0.05 level of significance (Steel et al.,1980).
RESULTS
Temperature and seed emergence
The average daily temperature for both air and soil (at a
depth of 5 10 cm) was below 15~C through early June, and remained around
20~C until mid-July. By August, the temperature start to drop until it reached
10~C by the end of September (Zhang et al.,1995a).
CA 022~737l l998-l2-04
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69
Experiment 1
The microbial densities associated with the root and in the
soil (rhizosphere) of the soybean plants are presented in table 12. The data
indicates differences among the PGPR strains. PGPR 2-68 showed more cfu
5 g~1 root in the first sampling than PGPR 1-102, while there was no significantdifference between the two PGPR at the second sampling at the
nonfumigated site~ PGPR 2-68, which had higher population densities on the
root, also showed higher microbial activities in the soil (rhizosphere) than
PGPR 1-102. There was no difference between the two PGPRs in the cfu 9-1
10 root or cfu 9~' soil at the second sampling of the nonfumigated site (Table 12).
CA 02257371 1998-12-04
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.E o ~ ~ oo oo N (D (~ 1~ ~ 1~ N
) N ~) ~t N N ~ O O
nl X ~~1:
~ _ t
_ ~ O
_ ~ 3 N N ~ 0~ ~ N C~ ~D 0
V ~ ~ O O O) ~ 1~ U') ~ I' t~ C'~l ~
, al o , ~ ~ ~ ~Y) ~ ~ ~ ~ o o
O _ G ~
-- cn ,
O~
~ ' O. ~ O Ln ~ CD u~ O O
-- ~ ~~ OC~ If~ N N ~ ~ ~~ CO O 00
~, ~o
~ ' ~N oo 1'- ~ ~ O 00 0 ~ CS~ ~)
E E ~a~ 01~ N ~ ~ ~
~ ~
m
~ ~ ~ - 3 o 3 o a r~3
---~ E o ~
_ E ~ a () c~
c s
O 3
o
.~_
~ ~ N J J
S~ ~ alll~TE S H EET (RULE 2~)
CA 02257371 1998-12-04
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70a
a~ -
z ~ Z * ~ ~ .c z
~-.E O ~ s
0 ~ ~n . tn o .
~ a ,~ a~ c ~ >~
w Q ~ ~C ~ ~ z ~ ~ ~ E Q ~
'' W ~ ~ ~n ~ n
3 ~ o E
o 4- -- E w
0 a, o ~ ~
~ E ~ ~ Z Z Z Z Z Z o o o
s ~O z cn ~ O
~ tn ~C ~~
O ~ E =
n
_ ~ >' l 'tn
~ .C C~ ' V
~ 0 Q c ~
.~ E ~ ~ _ ~
~, c E c "
E.~ ~ ~ tn tn
o 3 cc
o ~ 3 E ,~
1 ~ ~ ~ 1 1 1 ~ ~n ~
SlJesS 111 lJTE SHEET (RULE 26)
CA 022~7371 1998-12-04
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At the fumigated site, the same general pattern was
observed as PGPR 2-68 showed higher cfu 9-' root in both the first and
second sampling than PGPR 1-102 which indicate that this strain was able
to multiply and colonize the plant roots more effectiently than PGPR 1-102.
5 There was no difference between the two PGPR for the number of cfu g-' root
or cfu g~' soil at the second sampling (Table 13). There was no difference
observed between the two soybean cultivars, AC Bravor and Maple Glen, at
both fumigated and nonfumigated sites (Tables 12 and 13).
CA 02257371 1998-12-04
WO 98/44802 PCT/CA98/00336
~ E
X . ~ a~ ~) ~ ~ I~ O ~ ~ 0~
O O
_ ~ C
U '_ ~
N ~ ~ CD 1~ ) CD N
~ N ~ Ir~ ~ U~ 00 ~t U-) 00 ~
~ a. ~5~c ~ ) ~ ~ ~ ~ ~ O
~_ O ~
_ _.
_
.~ o
E o ~ ~ ~ ~ o ~ o a~ o
~ ~ ~ CO U~ N ~) 0~ ~D ~) N ~
2' ~ E
" E ~' o
E o ~, N
c 1~ ~ o u~
-- o) O ~ u~ ~ o o
~n.~
m ~ ~
~ ~ ~' m ~m c,m ~,
E ~
~ _
o~ C o o
~ ~ Q ~ ~ ~
~, cn
' n~
._ ._
~E ~
._ s
o,
o o
a~ .
'J
0 00 ~ ~
N J J
SU~ 111 UTE SHEE~ (RULE 25)
CA 02257371 1998-12-04
W O 98/44802 PCT/CA98/00336
72a
C C ,C~_ U
0 ' rJ
_ a ~ ~ z z z z z0 0 _
- ~ E ~ E ~ 0
~ ~ * ~ E u
~ 5 ~ O
~ ~ _ E ~O ~
_ ~ o ~ ~ 0
_ 3 -- ~z z Z Z Z Z ~ a~ ~
~ ~ ~ ~ --
E ~ * * cn (n ~ * ' r
_ ~ ~ E ~
~ ~ ~D ~
,_ ~ a.
m O ~ E ~
( ) c ,
a)
.~, c c
N ~ $
~ E c '--'
.
C 'r
~) ~cri ~ .c~ ,~
E ~ ~ 1 oJ
~ ~ a
8 ~ ~ . r ~ c *
s ~ a ~ ~ Q ~ E
: Q ~ ~ 0 *
Q ~ ~ ~ ~ ~ 1 ~ _ Z
SU~ TE SHEET(RULE 26)
CA 022~7371 1998-12-04
WO 98/44802 PCT/CA98tO0336
At the nonfumigated site, the population density of both
PGPR 2-68 and 1-102 on the root and in the soil had decreased by the
second sampling, except in the case of PGPR 2-68 co-inoculated with B.
japonicum onto soybean cultivar AC Bravor, where the population density
5 remained very high both on the root and in the soil at the nonfumigated site
~Table 12). Conversly, the population density of both PGPR 1-102 and 2-68
increased by the second sampling both on the roots and in the soil at the
fumigated site.
Experiment 2
There was no difference in the number of cfu g~' root at the
first sampling between PGPR 2-68 and 1-102, howerver, at the second
sampling PGPR 2-68 inoculated directly onto soybean cultivar AC Bravor had
the highest population density (Table 14). There was no difference in the
number of cfu g~' soil between the first and second sampling for either PGPR
15 (Table 14). There was not a great reduction in the population density of either
PGPR associated with the root between the first and second sampling.
CA 02257371 1998-12-04
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74
~ o
' 0 ~ ~ ~ 0 0 ~ n o
7~ 6 ~ ~ ~ ~7 0 o z z z ~ ~ ~,~
o E C O
c~ ~ ~ ~ ~ ~ 0 ~~ ~n
c 0 tO ~ N N N C/~ " E n
O ~ O ~ ~ ~ ~ ~ O O ~ Z ~ n ~n 1'
~ n--
o ~ ~ ~ ~ ~ ~ o
~) ~ U~ (D CO ~) 0 ~ (n--
~ _ 2~ 6 u~ u~ CD ui o o ~ Z
_ E o ~ ~ ~
E ~ ~ ~ ~D ~ ~ ~ r- o - ~n o
C~ O ~D CD OD C~ N 0 0 C/) ~t) C./) C
~ ~ ~ ~~ ~ ~ ~ o ~ Z Z Z ~ E ~
~ ~ O ~
E a~ n
Ç~ w m ~
_ ~ ";
o ~ N 1~ e:
~E c F ~n ~c
2 ~, Y ~
E 1 ~
~nn o.C
S '~ s ~ C
o c~ ~ ~ ~ ~ = "
SIJ~ 111 ~ITE SHEET (RU~F 26)
CA 022~7371 1998-12-04
W 098/44802 PCT/CA98/00336
At the fumigated site, there were more cfu 9-1 root for
PGPR 2-68 than PGPR 1-102 at the first sampling, while there was no
difference between the two PGPR at the second sampling. PGPR 2-68
inoculated onto AC Bravor plants had higher population densities in the soil
5 (rhizosphere) than PGPR 2-68 inoculated onto Maple Glen plants. There was
no difference between the two PGPR in terms of the number of cfu g~' root
or cfu g~' soil at the second sampling at the fumigated site (Table 15). There
were increases in the population densities for both PGPR associated with the
root and in the soil (rhizosphere) by the second sampling (Table 15).
CA 02257371 1998-12-04
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76
~ ,c o
C ~n . 1~ ~ 1--oo ~
~n ~) 6~ ~ ~ 'J N ~ z z Q
C~ o ._ F _
~ o ~
O . o ~ ~) ~ ~ ~ O O Z ,~ Z ~ ~ r
C ~ n
o a) o
- O ~ n ~ ~
O X 2~ I~ ~ o ~ ~ Z ~ n ~
E ~ ~ ~ ~ o CD ~ a~ ~ Z C o
~ 7 ~ ~ ~Z Z tD E ~
~ ~" U
m ~ ~ Q C~ :
~- O ~ 6 o
~ o O ~' E
E ' o ~D. tD ~ " D
U 1~ c, = N ~ C
,~ ~ .~ C
D ~ ~
.C ,cn
E F ~" o
~ 8 ~D
~ tD ~ ~
O F 1 _
O '~ o ._
S tD ~~ #
'~ s C C
Q C~ E
SU~a 1 l l UTE SHEET tRULE 26)
CA 022~7371 1998-12-04
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DISCUSSION
Plant growth-prun,oli,lg rhizobacteria strains S. Iiquefaciens
2-68 and S. proteamaculans 1-102 were selected following previous
controlled environment studies (Zhang et al., 1996b) in which nine P(~;PR
5 strains were tested for effects on soybean plant growth, development,
nodulation and nitrogen fixation over a range of RZTs under controlled
environment conditions. S. Iiquefaciens 2-68 was shown to perform well at
an optimal RZT (25~C), while S. proteamacuians 1-102 performed best at
suboptimal RZTs ranging from 18 to 15~C.
Colonization of soybean plants varied among PGPR strains
and soil conditions. In experiment 1, at the nonfumigated site, PGPR 2-68
colonized soybean plant roots more efficiently than PGPR 1-102 at the first
sampling, while there was no difference by the second sampling which
indicates that PGPR 2-68 was able to grow and colonize the root more
effectively initially but over time PGPR 1 -102 was able to grow and colonize
the roots as effectively as PGPR 2-68. PGPR 2-68 was able to proliferate
successfully in the soil (rhizosphere) as indicated by high cfu values at both
samplings.
Another interesting observation is that the population
densities of both PGPR 2-68 and 1 -102 with the different combinations of B.
japonicum and soybean cultivars had decreased in the second sampiing as
compared to the first, except for the combination of PGPR 2-68, B. japonicum
strain USDA110 and soybean cultivar AC Bravor where the population
density had increased in the second sampling compared to the first at the
nonfumigated site. These observations indicate that PGPR 2-68 cells can
survive and colonize the roots of the soybean plants effectively in the
presence of other soil microflora elements, and that they can tolerate the
change in field conditions, including temperature, over time without
reductions in population density.
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78
At the fumigated site, where assumely, no other microflora
compete with the PGPR, PGPR 2-68 showed the same pattern as in
nonfumigated conditions. In addition, the population density of both PGPR
had increased by the second sampling as compared to the first. These
observations suggest that both PGPR were able to survive and increase in
number in the absence of other ,-,icronora that would normally compete with
them and reduce their population density.
The results of experiment 2, in the absence of B.
japonicum, were different from those observed in experiment 1. There was
no difference between the two PGPR in the number of cfu g~' root at the first
sampling, while at the second sampling the combination of PGPR 2-68, B.
japonicum USDA110 and AC Bravor had the highest number of cfu g~' root.
There was no difference in the cfu g~' soil between the two PGPR strains at
both samplings of the nonfumigated site.
At the fumigated site, in experiment 2, the same pattern
was seen as in experiment 1. PGPR 1-102 also had more cfu g~' of both
roots and rhizosphere soil. There was no difference in the number of cfu g~1
root or cfu g-1 soil between the two PGPR at the second sampling for both
experiments.
Root colonization by introduced bacteria is considered as
an important step in the interaction of beneficial bacteria with the host plant.A rapid growth rate was suggested to be an important characteristic for
successful rhizosphere colonization (Rovira et al.,1983; Schorth et al.,1986).
De Weger et al. (1987) suggested that non-motile mutants colonize the roots
less effeciently than the corresponding wild types, while others found that
non-motile mutants and the corresponding wild types do not differ in their
colonizing ability (Parke et al., 1986; Scher et al., 1988). Chemotaxis of
bacteria to exudates was reported (Reinhold et al.,1985; Scher et al.,1985),
but the direct relationship between chemotaxis and successful colonization
, . . ~ . , ,
CA 022~737l l998-l2-04
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79
remains unclear. Movement along the root was also reported to be very
important for a successful root colonization (Chao et al., 1986; Schippers et
al., 1987). Adherence has also been suggested as an important feature for
rhizospheere competence and survival (Schippers et al., 1987; Vesper,
1987). Cells of bacteria in the genus Serratia are motile (Prescott, 1993).
Fluorescent pseudomonads, isolated from the crop
rhizosphere, are characterized as a highly rhizosphere competent as they are
capable of root colonization. This accounts for their predominance among the
PGPR. Several traits of the pseudomonads aid them in seed colonization,
such as higher cell division and motility (Arora et al., 1983; Scher et al.,
1985). However, these traits may not be directly relevant to subsequent root
colonization. For example, Howie et al. (1987) found that three nonmotile
mutants of P. ffuorescence colonized wheat roots as effectively as their
motile parents. Flourescent pseudomonads are able to establish high
population densities in the rhizosphere (Bahme et al., 1987; Suslow, 1982),
an important characteristic for the production of consistent plant growth
responses (Bahme et al., 1987; Klein et al., 1990; Kloepper et al., 1980 and
1985; Kloepper et al., 1991; Parke, 1991; Suslow, 1982). In general, there
has been little investigation of species in the genus Serratia as potential
PGPR.
Studies on rhizosphere colonization have been reviewed
recently by van Elsas et al. (1990), Kloepper et al. (1992) and Kluepfel
(1993). Van Elsas et al., (1990) reported that lack of consistent effectiveness
of the inoculant prevent successful application of PGPR strains in to the soil.
This always related was to ineffective colonization of the plant, as well as
poor survival and/or low activity of the introduced population. Xu et al. (1986)and Bull et al. (1991), demonstrated a positive relationship between root
colonization by a PGPR strain and disease suppression, suggesting that
methodologies which improve root colonization may also improve the
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performance of a PGPR strain in soil. The extent and amount of root
colonization needed by a PGPR strain to increase piant growth rely on
numerous interrelated factors. The choice of methods used to try to increase
rhizosphere colonization and plant growth has to take these factors into
5 consideration (Stephens, 1994).
Hebbar et al. (1992) reported that the colonization and
spread of Pseudomonas cepacia (which acts as a bio-control agent against
Fusarium moniliforme) on the roots and in the rhizosphere of maize depends
on the amount of inoculum on the seed. However, this was not a universal
10 observation. For example, the colonization of introduced pseudomonad
strains on maize (Scher et al., 1984) and wheat (Juhnkle et al., 1989) was
shown to be independent of the initial inoculum level. It is obvious that under
certain conditions, increasing the level of inoculum could increase the
rhizosphere competence of some, but not all, bacteria.
In a previous study, we found that co-inoculation with
PGPR and B. japonicum improved plant growth, development, yield
components, and final grain and protein yield under field conditions at both
fumigated and nonfumigated sites. Also application of PGPR with the B.
japonicum directly onto the seeds in the furrow at the time of planting also
20 improved plant growth at the fumigated site. The effects of PGPR S.
Iiquefaciens 2-68 and S. proteamaculans 1-102 on plant growth,
development, and final protein yield were shown to be not different, which
was attributed to variations in field soil temperature during the entire soybeangrowing season. In addition, a recent study at McGill university found that
25 co-inoculation of PGPR and B. japonicum accelerated the processes of
soybean nodulation and the onset of nitrogen fixation under short season
conditions .
Zhang et al., (1 996b), in a controlled environment
experiment, showed that both S. Iiquefaciens 2-68 and S. proteamacutans
- CA 022~737l l998-l2-04
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81
1-102 stimulate plant growth, development, and plant photosynthesis. At an
optimal RZT (25~C) S. liquefaciens 2-68 was reported to increase plant leaf
development and dry matter accumulation, while at 1 5~C RZT S.
proteamaculans 1-102 increased plant dry matter accumulation.
In summary, the results of this study indicated that PGPR
2-68 was able to grow and survive better than PGPR 1-102 under short
season conditions. A previous work, has shown that the combination of
PGPR 2-68 with AC Bravor plants had increased leaf area, seed number,
and grain protein yield suggesting that there is a relationship between the
ability of these PGPR to colonize the roots of the soybean plants and their
ability to stimulate soybean nodulation, nitrogen fixation, plant growth and
physiological activities under short season conditions.
EXAMPLE 4
Effect of PGPR and Kinetin on plant growth development, nodulation
and yield of soybean grown under short season conditions
In order to test whether the effect of PGPR could be further
modulated by the addition of additional factors, field experiments were
performed. Briefly, the experiment was conducted in 1997 and involved two
soybean cultivars (Maple Glen and Bayfield), three kinetin concentrations (0,
0.1 and 5 lum), and in the absence or presence of PGPR strains (1-102 or
2-68). Soybean plants were harvested to determine plant growth
development and final grain yield at one month after emergence, the early
podding stage, and final harvest. The results of this study are summarized
below as follows:
One month after emergence
The application of 5 ,um kinetin increased total
aboveground dry matter biomass per soybean plant by 12.5% when
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82
compared to the control (P=0.05). There was no difference in dry mater
biomass between the two kinetin concentrations (Figure 1).
Compared to 0.1 ,um kinetin, the application of 5 ,um kinetin
produced 54 and 83% more nodules and nodule weight per plant; and 66 and
5 87% more nodules and nodule weight per plant, respectively, were produced
than the control (Figures 2 and 3). The PGPR strain 1-102 with the 5 ,um
kinetin treatment showed the highest nodule number per plant while it had
the lowest nodule number at 0.1 1~ m kinetin (P< 0.06) (Figure 3).
Early podding stage
The application of 0.5 IJm kinetin resulted in 47 and 62% more nodules per
plant than the 0.1 ,um kinetin and the control treatment, respectively. Nodule
number was not different from the 0.1 ,um kinetin and control treatment on
either cultivar (Figure 4). The two soybean cultivars responded differently to
the kinetin concentrations in terms of nodule weight and root weight per
plant. This finding corroborates the well known findings that the genotype of
the cultivar affects its response to external stimuli (i.e. nitrogen fertilizers and
the like). Bayfield with 5 ,uM resulted in the highest nodule weight when
compared to its with 0.1 ,uM. (Figure 5). Under the 5 ~M kinetin
concentration, Maple Glen had the lowest root weight per plant while Bayfield
had the highest (Figure 6).
At final harvest the following were recorded
The application of kinetin resulted in 5.2 cm taller plants
than the control treatment over the two cultivars (Figure 7). There was no
difference in plant height between the two kinetin concentrations. Kinetin at
0.1 ,um had the lowest 100 seed weight when compared to kinetin at 5~Jm
and the control. One hundred seed weight was not differ from kinetin at 5 ~um
and the control (Figure 8). Bayfield was taller, had more total aboveground
dry matter biomass per plant, more seeds and more pods per plant than
Maple Glen, but had lower 1 00-seed weight than that of Maple Glen (Figures
. . .
CA 022~7371 1998-12-04
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83
7 and 9). Bayfield with 5 ,um kinetin resulted in the highest seed number per
plant while Maple Glen had the lowest (P<0.07) (Figure 10). PGPR 1-102
with 5 ,um kinetin showed the highest grain yield while the lowest was without
kinetin application and the same PGPR strain (Figure 11). However, the grain
5 yield was not different from the combination of PGPR 102 and the 5 ,um
kinetin treatment and the control (no PGPR and kinetin addition).
In summary, one month after emergence, soybean plant
growth, nodule formation and development increased with the application of
the highest kinetin concentration; the combination PGPR strain 1-102 with
10 0.5 ~Jm resulted in a positive response for nodule development. At the early
podding stage, the application of 0.5 ,um kinetin promoted nodule formation
but not plant growth; Bayfield with 5 ~m kinetin produced the highest root
weight. At final harvest, the application of kinetin resulted in taller plants;
Bayheld with 5 I~m kinetin resulted in the greatest number of seeds per plant
while the combination of PGPR 1-102 with 5 ~m kinetin resulted in more
grain yield than the same PGPR without kinetin.
Although the present invention has been described
hereinabove by way of preferred embodiments thereof, it can be modified,
without departing from the spirit and nature of the subject invention as
20 defined in the appended claims.
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Bahme et al., 1987, Phytopathol. 77: 1093-1100
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Bhuvaneswari et al., 1980, Plant Physiol. 66:1027-1031
Bull et al., 1991, Phytopathol. 81 :954-959
Burr et al., 1984, Crit. Rev. Plant Sci. 2:1-20
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