Inoculant production and formulation

J.H.G. Stephens
*
, H.M. Rask
MicroBio RhizoGen Corporation, 3835 Thatcher Avenue, Saskatoon, Saskatchewan, Canada S7R 1A3
Accepted 16 December 1999
Abstract
The formulation and commercial production of any Rhizobium or Bradyrhizobium legume inoculant requires the integration
of physical, chemical and biological parameters leading to both high target organism populations and long term survival of
these organisms over time at less than optimum conditions. To achieve this, all raw materials processing and bacteriological
needs must be addressed, and quality products developed within narrow budgetary constraints. It is only through an
understanding of the needs and limitations of both the products physical and Rhizobium/Bradyrhizobium requirements that an
effective and efcient legume inoculant can be commercially produced. # 2000 Elsevier Science B.V. All rights reserved.
Keywords: Rhizobium; Inoculant production; Peat and granular formulations; Strain selection
1. Introduction
Rhizobium/Bradyrhizobium inoculants have been
used for over a century but research directed toward
optimizing their impact is an ongoing activity. Smith
(1992), and Brockwell and Bottomley (1995), have
reviewed the history of legume inoculant production
and desirable characteristics of an inoculant; Thomp-
son (1980) and Lupwayi et al. (2000), included aspects
on quality control relating to these products. At times
the research ndings on inoculants are inapplicable at
the eld level or impracticable for the manufacturer or
end user levels. One of the greatest barriers to pre-
dicting, and consistently achieving, responses to
inoculation is the lack of knowledge relating to target
organism fate over time and under commercial crop
production conditions.
The end user of a legume inoculant is primarily
concerned with crop productivity, not bacterial phy-
siology nor ecology. Inoculant manufacturers must
accept this reality and ensure that the probability of
successful inoculation is maximized, even though the
end user may not understand the bacteriological prin-
ciples on which the product is based. Since crop
response to inoculation, in most instances, is the result
of appropriate strain selection, and target organism
population (Muldoon et al., 1980; Amarger, 1981;
Brockwell et al., 1988; Hume and Blair, 1992; Fes-
enko et al., 1995), a successful inoculant can only be
formulated through a process of addressing the known
product substrate variables that affect target organism
viability. Regardless of the formulation of any pro-
duct, nal product quality must be assured through a
Field Crops Research 65 (2000) 249258
*
Corresponding author. Tel.: 306-373-3060; fax: 306-374-
8510.
E-mail address: stevestephens.mbrsask@sk.sympatico.ca (J.H.G.
Stephens).
0378-4290/00/$ see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S0 3 7 8 - 4 2 9 0 ( 9 9 ) 0 0 0 9 0 - 8
process of strict quality assurance. Quality assurance
as opposed to quality control requires that every step
in the production process have set limits and only if
these limits are met, or surpassed, can further proces-
sing toward a nished product be undertaken.
The primary benet of promoting biological nitro-
gen (N
2
) xation in grain and forage legumes is the
enhancement of yield potentials without the use of
exogenous N fertilizers. This ability to promote N
2
xation has resulted in commercial inoculant produc-
tion facilities in numerous countries and on every
continent (Brockwell and Bottomley, 1995). Unfortu-
nately, the widespread availability of legume inocu-
lants has not resulted in international standards of
quality, nor of rates of use for these products. Many
countries, including Australia, the Netherlands,
Rwanda, Thailand and Russia (Smith, 1992; Marufu
et al., 1995), legislate requirements for minimum
populations of target organisms and contaminants
per unit weight of product. Canada and France legis-
late product quality by dening the number of viable
Rhizobium/Bradyrhizobium required per seed, or a
rate of application, for a full range of crops. The
United States and United Kingdom leave product
quality and rates of application to the discretion of
the manufacturer (Smith, 1992). This lack of inter-
nationally accepted regulations for legume inoculant
quality and usage parameters has led, in many
instances, to inadequate performance of inoculants
and to the subsequent abandonment of their use.
The production of effective legume inoculants can,
however, partially overcome inadequacies in legisla-
tion with the manufacturer taking responsibility for
the design and implementation of steps leading to a
consistently high quality and effective product.
2. Formulation of inoculants
Formulation inadequacies are often the most com-
mon barriers to the commercialization of legume
inoculants. A micro-organism may function optimally
under laboratory conditions, but formulating that
organism into an end-user affordable product that is
consistently able to bring about equivalent results
under eld conditions is a difcult step. Once an
inoculant formulation which works in situ has been
developed, it must be rened to allow for the sophis-
tication of the end-user, for instance, the level of
mechanization used by the differing farm commu-
nities and the time frames available during the busy
seeding period.
Inoculants are usually commercialized in one of
three forms; powder in the form of processed sedge
peat moss, liquid and granular. Most granular prepara-
tions are in the form of a peat prill though, more
recently, hard mineral based products have been devel-
oped. Powdered products are usually used for seed
inoculation and applied directly to the seed. Liquid
products may be applied directly to the seed or placed
in the furrow during planting. Granular products can
be placed directly in the furrow, deep banded below
the seed or side banded and in the latter instance
preferably deeper than the seed. Peat our continues
to be the most popular method of delivery for Rhizo-
bium/Bradyrhizobium inoculants and has been since
1895, the historically accepted date of initiation of
legume inoculant production and use. Liquid and
granular alternatives are available in limited quantities
in North America and Europe.
Peat our prepared from sedge, as opposed to
sphagnum, peat moss, is most commonly used in
the production of legume inoculants. The producer
then has the choice to sterilize or not to sterilize the
processed peat our prior to the introduction of the
rhizobia. Sterile peat our substrates normally display
higher populations than those that are non-sterile and
an enhanced shelf life (J.H.G. Stephens, unpublished).
A positive advancement in peat inoculants in recent
years has been the incorporation, in some instances, of
a sticking agent: this has not only made the product
more user friendly but has also resulted in enhanced
uniformity of coverage.
The liquid inoculants currently available display
various broth formulations, each the result of indivi-
dual producer's research and development activities.
Liquid products have been promoted as being easier to
handle (Hynes et al., 1995), and in many instances
display efcacy equivalent to peat products. However,
in many instances their limited shelf life and cool
temperature storage needs increase handling and end
user costs in developed countries and precludes their
use in most developing countries. A range of other
formulations including oil based products (Kremer
and Peterson, 1983a,b; Hoben et al., 1991), have also
been studied but there has been little move toward
250 J.H.G. Stephens, H.M. Rask / Field Crops Research 65 (2000) 249258
their commercialization. Production of Rhizobium/
Bradyrhizobium concentrates, which would be diluted
with water before use, have also been developed but
maintenance of organism populations within the con-
centrates over time has been problematic.
Recently, there has been increased interest in the
development of granular inoculants (Fraser, 1975;
Hedge and Brahmaprakash, 1992; Fouilleux et al.,
1994, 1996). Crop nodulation and yields achieved
with granular preparations can be equivalent to those
achieved with peat, liquid products and liquid seedbed
inoculants (Bezdicek et al., 1978; Brockwell et al.,
1980; Dubetz et al., 1983; Rice and Olsen, 1992). Such
granular inoculants are particularly suitable to farming
systems in developed countries where the seeding
equipment commonly includes multiple, seed, fertili-
zer and inoculant delivery systems.
A major advantage of the granular inoculants is the
ability to control placement and application rate as
needed. Although further evaluation is needed, current
indications are that best results are obtainable through
deep banding either directly below the seed or to the
side, as opposed to in the seedbed. Seed inoculants
have several disadvantages not found with granular
inoculants (Brockwell et al., 1980). Amajor one is that
each seed can carry only a limited amount of inoculum
and therefore number of bacteria. For small seeded
species, such as white clover, this means that each seed
receives approximately 10
3
rhizobia. This can con-
strain inoculant success in unfavorable environments
or where soils contain indigenous rhizobia. There is
also the potential for inoculant loss as occurs in crops
displaying epigeal germination. Further, seed applied
inoculants are inappropriate for those crops with
delicate seed coats, for instance peanut, and those
species releasing bactericidal compounds from the
seed. Another disadvantage of seed applied inoculants
is where seed treatment with fungicidal or insecticidal
chemicals can be detrimental to rhizobial survival.
(Brockwell et al., 1980; Fouilleux et al., 1994). The
bulk of these seed applied inoculant difculties can be
overcome through the use of granular preparations
since the target organisms are not in direct contact
with chemicals that could be detrimental to their long
term survival.
Granular inoculants also appear to increase N
2
xation through improvement in nodulation patterns
(Hardarson et al., 1989). Granular inoculants placed
either directly below or below and to the side of the
seed, encourage lateral root nodulation as opposed to
crown nodulation obtained with seed applied products.
Lateral root nodulation was reported to enhance N
2
xation over the growing period of soybean. Hard-
arson et al. (1989) and McDermott and Graham(1989)
also noted that placement of inoculant at time of
seeding was relevant and that optimizing placement,
as opposed to seed application, resulted in enhanced
nodule occupancy of the desired strain. Although N
2
xation within a crop can be enhanced through place-
ment of a granular inoculant, agronomy trials designed
to dene optimized placement for all crops must
continue.
Although a dry, solid core, granular product has
only recently been marketed, a compressed peat, or
prill, product has been available for several decades. In
the former product the difculty has been to obtain a
product displaying a low water activity while main-
taining acceptable levels of rhizobia during the shelf
life of the product. Both granular products display
acceptable ow characteristics when applied through
large scale seeding equipment.
Alternative formulations continue to be investi-
gated. These include lyophilized cultures, cell suspen-
sions in oil and cell concentrates. The major areas of
investigation, however, revolve around increasing rhi-
zobial populations per unit weight or volume of
product, organism efcacy and product durability as
this relates to product shelf life at the retail level.
3. Inoculant production
The three concerns in inoculant production are the
quality and processing of the carrier, the purity and
efciency in nodulation and N
2
xation, of the culture,
and achieving adequate cell numbers in broth culture
and nished product.
3.1. The carrier
Smith (1992), noted that the raw materials chosen
for use in conventional, seed applied, inoculants are
determined by availability, consistency of quality and
cost. The carrier must display two fundamental prop-
erties; it must support growth of the target organism
and maintain desired populations of inoculant strains
J.H.G. Stephens, H.M. Rask / Field Crops Research 65 (2000) 249258 251
over an acceptable time period. To achieve these goals
a carrier must also display high water holding capacity
and retention characteristics, display chemical and
physical uniformity and be non-toxic to inoculant
strains and be non-toxic to inoculant strains and
environmentally safe. It needs also to permit growth
after introduction of the rhizobia, have an acceptable
pH, rapidly release organisms upon use and be in
abundant supply. Most sedge peats fulll the bulk of
these requirements however the supply of this peat is
becoming problematical in many instances.
Processed peats can be sterilized prior to the addi-
tion of rhizobia or left non-sterile. Although some
producers continue to use non-sterile peat, Roughley
and Vincent (1967), noted that sterile carriers gener-
ally support higher populations and display much
longer shelf lives. Further, the use of a sterile peat
carrier signicantly reduces threats to quality resulting
from the presence of contaminants. Sterile peat also
lowers costs of culture production by extending the
broth, through culture dilution, while still achieving a
higher nal population density. The disadvantages of
sterilizing the peat carrier are the cost of sterilization,
identifying a sterilization unit with sufciently large
and rapid throughput capacity and the need to follow
aseptic procedures during culture injection (Smith,
1992). Nuclear, or g, irradiation has historically been
the sterilization method of choice but is expensive and
slow. Autoclaving has been used in laboratory envir-
onments but is laborious, costly and time consuming
and in a commercial context can result in inoculant
quality compromised by the production of toxins
during sterilization. Electron acceleration is the most
recent technology used for sterilization. This process
is non-nuclear and depends upon the exploitation of a
series of acceleration cavities which ultimately result
in an electron beam which generates 1010
6
eV with
which the peat is irradiated, and hence sterilized. One
of the greatest benets of electron beam sterilization is
the turn-around time since the pre-packaged peat our
is only exposed to the sterilization process for seconds
versus hours for g irradiation.
Uruguay, a country in which sterile peat is man-
dated, is unusual in having peat deposits, but not
the facilities for routine peat sterilization. Instead,
pre-sterilized packaged peat is imported from Canada
and inoculated in Uruguay (C. Labandera, pers.
comm.).
Although peat is the carrier of choice, it is not
ubiquitously distributed and a more readily available
substrate may be required. Alternative carriers, both
natural and derived, have been investigated and
include mineral soils (Chao and Alexander, 1984),
plant byproducts (Sparrow and Ham, 1983), various
clays (Graham-Weiss et al., 1987), charcoal (Kremer
and Peterson, 1983a; Sparrow and Ham, 1983), lig-
nite, a range of coals (Paczkowski and Berryhill,
1979), composted plant materials (Kostov and Lynch,
1998), animal manure, perlite, rock phosphate, talc,
entrapped alginate beads (Bashan, 1986), polyacryla-
mide gels (Dommergues et al., 1979), and mixtures of
xanthan and carob gum (Jung et al., 1982). Often the
ability of an alternative carrier, or entrapment meth-
odology, to sustain organism growth is marginal while
cost, consistency of supply and quality, in many
instances, work against more widespread adoption
of these alternative materials.
In addition to peat prills, granular formulations have
included carbon amended clays (Fouilleux et al.,
1996), alginate/perlite beads (Hedge and Brahmapra-
kash, 1992), sand coated with peat inoculant (Cham-
ber, 1983), coated polythene beads and marble chips
(Brockwell et al., 1980). Although none of these are
known to have reached the commercial market, a peat
prill and a hard granular product are currently avail-
able on the North American market. The composition
of these two products are proprietary information.
Consistency is an absolute requirement in any
carrier substrate. Because of the integral part the
carrier plays in the production and ultimate quality
of the product, it is desirable that it undergo ``t for
use'' trials on receipt and prior to further processing.
Measurable criteria, be they physical, chemical or
biological, must be established and steps taken to
ensure that these criteria are met or exceeded with
each shipment of rawmaterials. If these criteria cannot
consistently be met, the carrier material will limit the
reliability of any formulation that is subsequently
developed. This is unacceptable and necessitates
mutual concern for quality by both the supplier and
the inoculant producer and the need for routine testing,
by the producer, of the received raw materials.
In virtually every instance product raw materials
must be modied prior to their use as a quality carrier.
In the case of bulk peat moss, the raw material must be
dried and ground into ne our. Thompson (1980),
252 J.H.G. Stephens, H.M. Rask / Field Crops Research 65 (2000) 249258
suggested that a particle size of less that 0.075 mm,
results in optimum surface coverage of seed per unit
weight of inoculant. Most peats are inherently acidic
and need to be neutralized and, in some instances, to
have a buffer added prior to substrate sterilization and
culture addition. Although most sedge peats are cap-
able of sustaining target organism growth and repro-
duction, sufcient to support values in the mid log 8
range, the addition of macro and/or micro nutrients to
the substrate to enhance population densities cannot
be dismissed.
3.2. Strain selection
To optimize nitrogen xation in commercially
important legumes, culture organisms need to be
competitive with the indigenous populations and be
efcient in N
2
xation upon nodule formation. Even if
competitiveness for available nutrients is one of the
strain selection criteria, soil rarely provides the rich
environment evident in most culture media. Pre-con-
ditioning, and hence competitiveness of the inoculant
strain can be achieved, in part, through the use of a
nutrient-limited medium. The use of such a medium
also reduces the likelihood of selecting a non-compe-
titive strain.
The development of an inoculant that can be used
for a range of crops of the same genus, and which
contains a cocktail of strains, as opposed to single
strain inoculants specic to a single crop, is a con-
tinuing subject of discussion. Regardless of any orga-
nizational preferences for a single or multi-strain
product, there is a need to select strains for specic
geographic, soil and environmental regions and, ide-
ally, for individual varieties of a given legume species.
The top performing strain(s) for a given market area
must be compatible with the commonly grown crop
cultivars in that area, and display the adaptability to be
effective and efcient over a range of geographic and
environmental conditions. This adaptability is an
obvious requirement since soil types, moisture pat-
terns and cultivar preferences can, and do, alter over
small geographic areas. Lynch (1983), stated ``Each
microbial species will have an optimum for each
physical or chemical factor and its growth, or activity,
will decline either side of that optimum, governing its
contribution to the total population.'' Further it has
been noted that microbial preferences can change
within macro and micro climates determined by soil
texture (Mahler and Wollum, 1981; Osa-Aana and
Alexander, 1982), structure, aeration, pH (Cassman
et al., 1981), temperature and available moisture
(Hardarson and Jones, 1979; Clark et al., 1986; Sprent
et al., 1986), nutrient status, organic matter content
(Semu and Hume, 1979), and weather. It is apparent,
therefore, that rhizobial activity can vary greatly with
environmental pressures, with clear differences in
response found both between and within species.
Selection of an appropriate organism is critical and
cannot be a process carried out over a single year of
eld trials and over few locations.
That different Rhizobium and Bradyrhizobium iso-
lates can vary in efciency with different cultivars and
environments was noted by Jones and Hardarson
(1979), Rennie and Kemp (1983a,b), Hobbs and
Mahon (1982), Kipe-Nolt et al. (1993) and Provorov
et al. (1994). Hardarson and Jones (1979) and Hynes
and O'Connell (1990), also found variability between
soils as a result of differing soil physical and chemical
conditions. This apparent strain selection, as a result of
soil differences, was also noted by Shishido and
Pepper (1990) and Sattar et al. (1995), who reported
that the most effective strains for a given crop, in a
given geographic region, were often strains isolated
from that region. The basis for such ecosystem
response has been a subject of considerable recent
study by microbial ecologists.
Competitiveness of selected strain(s) with indigen-
ous populations of rhizobia (Winaro and Lie, 1979;
Broughton et al., 1982; van Rensburg and Strijdom,
1982; Fesenko et al., 1995) is also a major factor in
strain selection. There is, therefore, a denite need for
any organization developing inoculants to have the
means of recovering and typing nodule occupants. To
merely use a strain displaying acceptable N
2
xation
in-vitro is not acceptable. The inoculant organism
must display both acceptable N
2
xation and competi-
tiveness, for the inoculant to be effective. In an attempt
to overcome strain/soil/cultivar difculties multi-
strain products have been investigated. There are
numerous difculties associated with the multiple
strain approach, not the least of which is attempting
to ensure that each strain is equally represented.
Frankenberg et al. (1995) indicated that this was
difcult to achieve because of differing growth rates
between strains of the same species. Caldwell and Vest
J.H.G. Stephens, H.M. Rask / Field Crops Research 65 (2000) 249258 253
(1968), Bailey (1988), Somasegaran and Bohlool
(1990), and others have also reported that crop N
2
xation, when inoculated with a multi-strain inocu-
lant, was generally less than that associated with a
single strain product. If nodule occupancy reects the
ratio of each strain in the inoculant, the N
2
xing
potential of a multi-strain preparation is likely to
reect the effectiveness and competitiveness of the
predominant strain or strains.
3.3. Culture production
Although the literature abounds with rhizobial
media formulations, these, almost without exception,
were developed for general laboratory procedures.
Under commercial legume inoculant production con-
ditions, attempts to condition the culture, to ensure
survival and reproduction in an environment where
nutrients are less readily available, must be made.
Although the development of, for instance, a carbon
limited medium is straightforward, there is also a need
to ensure that the medium can support populations at,
or exceeding, 10
9
CFU ml
1
. This value is being
targeted for the production of sterile peat based pro-
ducts since the mother culture is diluted, with sterile
deionized or reverse osmosis, water, to at least 1/50
and in some instances 1/250 prior to injection into the
sterile peat. For granular products the relationship
between mother culture population densities and n-
ished product quality is direct, the better the mother
culture the better the product.
By-products from food processing, cheese whey,
corn steep liquor, malt sprouts and malt bagasse have
been reported to be acceptable carbon and nitrogen
sources for rhizobial culture medium (Meade et al.,
1985; Bissonnette et al., 1986; Balatti et al., 1991).
The use of by-products from the food processing
industry can be problematic since few such ``waste''
products can assure continuity of quality. Even the use
of conventional medium substrates is fraught with
pitfalls. For instance Skinner et al. (1977) noted that
excessive use of yeast extract as a carbon and nitrogen
source resulted in both cell morphological changes
and decreased nodulation potential.
Along with growth medium constituents, and con-
stituent concentrations, fermentation variables such as
dissolved oxygen (DO
2
), temperature, pH and impel-
ler tip speed must be established for each organism.
Current fermenter technology can provide continuous
monitoring, and control of virtually all measurable
parameters and as such custom production of mother
cultures of any chosen Rhizobium/Bradyrhizobium
species or strain is possible and, indeed, desirable.
Commonly used DO
2
values are between 90 and
100%, and temperature values between 25 and 288C
are standard. Because fermenter starter cultures are
normally produced in swirl asks, where culture pH
and DO
2
are rarely controlled, the inclusion of a
buffer, such as MOPS (3-[N-Morpholino]propanesul-
fonic acid), should be considered. Where possible,
DO
2
values in the starter cultures should also be
controlled since normal values for this variable, in
swirl or stir asks, have been seen to be in the 2530%
saturation range for mature cultures. Lack of control
of these variables in starter cultures can result in
signicant lag phase on transfer of the mother culture
to the fermenter. This results in decreased population
densities at the time of scheduled harvest.
3.4. Finished product production
Production considerations must be scheduled to suit
employee work periods because a completely inte-
grated system must be developed. This must be amen-
able to specied dates of culture harvest at which the
culture always conforms to a set of measurable para-
meters.
It is only by developing a complete culture produc-
tion system, from agar slope or glycerol deep to swirl/
stir ask to fermenter, that nished product quality can
be assured. This assurance, in turn, requires a set of
legislated quality standards and in house minimums.
Ideally, in house standards should be in excess of
legislated standards and greater than those which have
been experimentally shown to result in enhanced crop
productivity.
3.5. Assuring a quality product
Quality Assurance ensures that, at each stage of the
production process, pre-dened criteria must be
met or the production run is aborted. Employing a
quality assurance approach to inoculant production
reects the integration of equipment performance, raw
material quality and other variables required for a
quality product. It also results in minimal, if any,
254 J.H.G. Stephens, H.M. Rask / Field Crops Research 65 (2000) 249258
loss due to a nished product not displaying accep-
table target organism populations and nodulation
potentials.
A quality assurance system guards against compla-
cency since product upgrading, as a result of techno-
logical advances or consumer demand, is integral to
the system. Changes must only benet the process and
therefore should only be brought about after discus-
sion and testing. If a proposed change does not
enhance the quality and dependability of the nished
product the change must not take place.
The production of a quality legume inoculant is the
coming together of a series of disparate activities and
apparently non-related processes. Ultimately, how-
ever, the disparate facets of production result in a
product displaying the potential to benet not only the
farm community but the producer and society in
general.
4. Co-inoculants
Co-inoculants, in this instance, are those which
contain rhizobia and organisms of other genera and
species. The requirements for production of these
inoculants are equivalent to those for rhizobia only
products but organism/organism interactions in these
products must be confronted.
It has been found (J.H.G. Stephens, unpublished),
during the development of co-inoculants containing
Rhizobium leguminosarum biovar viceae, a fungal
antagonistic Pseudomonas species and/or a growth
promoting Bacillus species that mixed population
products rarely reached individual organism cell
populations obtained for each organism in mono-
culture. This is evident in Table 1. As is also
evident from Table 1, in only one instance did co-
inoculated populations approach values evident in
the monoculture controls. This equivalency of
populations was only seen in a mix of RGPs7, a
pseudomonad, and RGP2, a R. leguminosarum biovar
viceae strain. Co-inoculants hold considerable pro-
mise, however care must be taken to ensure rhizobial
populations do not suffer when cultured with non-
rhizobia.
Indications are that rhizobial strain selection may
overcome some of the mixed culture competition
effects. The choice of selecting a rhizobial strain
for compatibility with a secondary organism, as
opposed to N
2
xation potential and competitiveness,
would be a partial option in the instance of the
Pseudomonas sp. noted in Table 1. Any such choice
will, however, require extensive eld testing to ensure
that any change in rhizobial strain does not result in
less than equivalent N
2
xation and crop productivity.
From this preliminary study it appears that the pro-
duction of co-inoculants will require extensive in-vitro
and in-situ investigations if the positive attributes
associated with each organism are to be effectively
exploited.
Table 1
Mean populations of three differing bacterial genera seven days post-injection into sterile peat our and when incubated at 278C. Injected
cultures displayed CFU values between log 9 and 9.4
Injected organism/organisms CFU log
10
injected organisms
RGPs7
a
RGB19 RGP2 RGC1
RGPs7 9.1
RGB19 9.7
RGP2 9.0
RGC1 9.3
RGPs7RGP2 9.3 9.0
RGPs7RGC1 9.1 8.4
RGB19RGP2 7.2 7.5
RGB19RGC1 7.5 8.6
RGPs7RGB19 8.8 7.3
RGPs7RGB19RGP2 7.4 7.6 7.7
RGPs&RGB19RGC1 7.9 7.3 7.8
a
RGPs7, Pseudomonas sp., RGB19, Bacillus sp., RGC1 and RGP2, Rhizobium leguminosarum biovar viceae strains.
J.H.G. Stephens, H.M. Rask / Field Crops Research 65 (2000) 249258 255
5. Summary
The development of an effective seed or soil applied
legume inoculant requires the integration of physical,
chemical and biological processes. With conventional
seed applied peat products, physical processing of the
raw product is required to achieve particle size dis-
tribution appropriate to the product, and chemical
changes may be needed to neutralize the normally
acidic peat. If the product is to be self-adhering, a non-
toxic sticking agent must be identied and critical
concentrations determined. Biological parameters
include the selection of an efcient and effective
rhizobia that display high population densities in
the processed, modied, peat our. All these processes
and manipulations must be completed against strin-
gent budgetary guidelines that allow the nished
product to be delivered to the end user at a cost-
effective price.
The process of development of any inoculant, peat,
liquid or granular, can be simplied down to its
individual steps, however no one step stands indepen-
dent from that which precedes or follows it. Each step
in the process, during the development stage, must be
seen to be compatible with prior and subsequent
activities. It is only through an appreciation of these
interdependent activities that a quality inoculant,
legume or otherwise, can be successfully developed.
References
Amarger, N., 1981. Selection of Rhizobium strains on their com-
petitive ability for nodulation. Soil Biol. Biochem. 13, 481486.
Bailey, L.D., 1988. Inuence of single strains and a commercial
mixture of Bradyrhizobium japonicum on growth, nitrogen
accumulation and nodulation of two early-maturing soybean
cultivars. Can. J. Plant Sci. 69, 41418.
Balatti, A.P., Alcaraz, M., Ronchi, A.L., Grassano, A., 1991.
Rhizobium and Bradyrhizobium biomass production in batch
culture with a malt bagasse medium. Rev. Lat-Amer. Microbiol.
33, 219223.
Bashan, Y., 1986. Alginate beads as synthetic inoculant carriers for
slow release of bacteria that affect plant growth. Appl. Environ.
Microbiol. 51, 10891098.
Bezdicek, D.F., Evans, D.W., Abede, B., Witters, R.E., 1978.
Evaluation of peat and granular inoculum for soybean yield and
N xation under irrigation. Agron. J. 70, 865868.
Bissonnette, N., Lalande, R., Bordeleau, L.M., 1986. Large-scale
production of Rhizobium meliloti on whey. Appl. Environ.
Microbiol. 52, 838841.
Brockwell, J., Bottomley, P.J., 1995. Recent advances in inoculant
technology and prospects for the future. Soil Biol. Biochem. 27,
683697.
Brockwell, J., Gault, R.R., Chase, D.L., Hely, F.W., Zorin, M.,
Corbin, E.J., 1980. An appraisal of practical alternatives to
legume seed inoculation: eld experiments on seed bed
inoculation with solid and liquid inoculants. Aust. J. Agric.
Res. 31, 4760.
Brockwell, J., Herridge, D.F., Morthorpe, L.S., Roughley, R.J.,
1988. Numerical effects of Rhizobium population on legume
symbiosis. In: Beck, D.P., Materon, L.A. (Eds.), Nitrogen
Fixation by Legumes in Mediterranean Agriculture. Martinus
Nijhoff, Dordrecht, The Netherlands, pp. 179193.
Broughton, W.S., Samrey, U., Bohlool, B.B., 1982. Competition for
nodulation of Pisum sativa cv afghanistan requires live rhizobia
and a plant component. Can. J. Microbiol. 28, 162168.
Caldwell, B.E., Vest, G., 1968. Nodulation interactions between
soybean genotypes and serotypes of Rhizobium japonicum.
Crop Sci. 8, 680682.
Cassman, K.G., Munns, D.N., Beck, D.P., 1981. Growth of
Rhizobium strain at low concentrations of phosphate. Soil Sci.
Soc. Amer. J. 45, 520523.
Chamber, M.A., 1983. Inuence of several methods for rhizobial
inoculation on nodulation and yield of soybeans. Plant Soil 74,
203209.
Chao, W.L., Alexander, M., 1984. Mineral soils as carriers for
rhizobium inoculants. Appl. Environ. Microbiol. 47, 9497.
Clark, K.W., Brockwell, J., Thompson, J.A., 1986. Role of
inoculants in improving nitrogen xation in legumes. In:
Summereld (Ed.), Abstracts of the International Food Legume
Research Conference on Pea, Lentil, Fababean and Chickpea.
Martinus Nijhoff, Dordrecht, The Netherlands, p. 16.
Dommergues, Y.B., Diem, H.G., Divies, C., 1979. Polyacrylamide
entrapped Rhizobium as an inoculant for legumes. Appl.
Environ. Microbiol. 37, 779781.
Dubetz, S., Major, D.J., Rennie, R.J., 1983. Production practices
for early maturing soybeans in southern Alberta. Can. J. Plant
Sci. 63, 641647.
Fesenko, A.N., Provorov, N.A., Orlova, I.F., Orlov, V.P., Simarov,
B.V., 1995. Selection of Rhizobium leguminosarum bv. viciae
strains for inoculation of Pisum sativum L. cultivars: analysis of
symbiotic efciency and nodulation competitiveness. Plant Soil
172, 189198.
Fouilleux, G., Revellin, C., Catroux, G., 1994. Short-term recovery
of Bradyrhizobium japonicum during an inoculant process
using mineral microgranules. Can. J. Microbiol. 40, 322
325.
Fouilleux, G., Revellin, C., Hartmann, A., Catroux, G., 1996.
Increase of Bradyrhizobium japonicum numbers in soils and
enhanced nodulation of soybean (Glycine max (L) Merr.) using
granular inoculants amended with nutrients. FEMS Microbiol.
Ecol. 20, 173183.
Frankenberg, C.L.C., Freire, J.R.J., Thomas, R.W.S.P., 1995.
Growth competition between two strains of Bradyrhizobium
japonicum in broth and in peat-based inoculant: dinitrogen
xation efciency and competition for nodulation sites. Rev.
Microbiol. Sao Paulo 26, 211218.
256 J.H.G. Stephens, H.M. Rask / Field Crops Research 65 (2000) 249258
Fraser, M.E., 1975. A method of culturing Rhizobium melilot on
porous granules to form a pre-inoculant for lucerne seed. J.
Appl. Bacteriol. 39, 345351.
Graham-Weiss, L., Bennett, M.L., Paau, A.S., 1987. Production
of bacterial inoculants by direct fermentation on nutrient-
supplemented vermiculite. Appl. Environ. Microbiol. 53, 2138
2140.
Hardarson, G., Golbs, M., Danso, S.K.A., 1989. Nitrogen xation
in soybean (Glycine max L. Merril) as affected by nodule
patterns. Soil. Biol. Biochem. 21, 783787.
Hardarson, G., Jones, D.G., 1979. Effect of temperature on
competition amongst strains of Rhizobium trifolii for nodula-
tion of two white clover varieties. Ann. Appl. Biol. 92, 229
236.
Hedge, S.V., Brahmaprakash, G.P., 1992. A dry granular inoculant
of Rhizobium for soil application. Plant Soil 144, 309311.
Hobbs, S.L., Mahon, J.D., 1982. Effects of pea (Pisum sativum)
genotypes and Rhizobium leguminosarum strains on N
2
(C
2
H
2
)
xation and growth. Can. J. Bot. 60, 2592600.
Hoben, H.J., Aung, N.N., Somasegaran, P., Kang, U., 1991. Oils as
adhesives for seed inoculation and their inuence on the
survival of Rhizobium spp. and Bradyrhizobium spp. on
inoculated seeds. World J. Microbiol. Biotech. 7, 324330.
Hume, D.J., Blair, D.H., 1992. Effect of members of Bradyrhizo-
bium japonicum applied in commercial inoculants in soybean
seed elds in Ontario. Can. J. Microbiol. 38, 582593.
Hynes, M.F., O'Connell, M.P., 1990. Host plant effect on
competition among strains of Rhizobium leguminosarum.
Can. J. Microbiol. 36, 864869.
Hynes, R.K., Craig, K.A., Covert, D., Smith, R.S., Rennie, R.J.,
1995. Liquid rhizobial inoculants for lentil and eld pea. J.
Prod. Agric. 8, 547552.
Jones, D.G., Hardarson, G., 1979. Variation within and between
white clover varieties in their preference for strains of
Rhizobium trifolii. Ann. Appl. Biol. 92, 221228.
Jung, G., Mugnier, J., Diem, H.G., Dommergues, Y.R., 1982.
Polymer-entrapped Rhizobium as an inoculant for legumes.
Plant Soil 65, 219231.
Kipe-Nolt, J.A., Vargas, H., Giller, K.E., 1993. Nitrogen xation in
breeding lines of Phaseolus vulgaris L. Plant Soil 152, 103
106.
Kostov, O., Lynch, J.M., 1998. Composted sawdust as a carrier for
Bradyrhizobium, Rhizobium and Azospirillum in crop inocula-
tion. World J. Microbiol. Biotech. 14, 389397.
Kremer, R.J., Peterson, H.L., 1983a. Effects of carrier and
temperature on survival of Rhizobium spp. in legume inocula:
development of an improved type of inoculant. Appl. Environ.
Microbiol. 45, 17901794.
Kremer, R.J., Peterson, H.L., 1983b. Field evaluation of selected
Rhizobium in an improved legume inoculant. Agron. J. 75,
139143.
Lupwayi, N.Z., Olsen, P.E., Sande, E.S., Kayser, H.H., Collins,
M.M., Singleton, P.W., Rice, W.A., 2000. Inoculant quality and
its evaluation. Field Crops Res. 65, 259270.
Lynch, J.M., 1983. The soil as a habitat for micro-organisms. In:
Soil Biotechnology: Microbiological Factors in Crop Produc-
tivity. Blackwell Scientic, Oxford, London, pp. 524.
Mahler, R.L., Wollum, A.G., 1981. The inuence of irrigation and
Rhizobium japonicum strains on yields of soybeans grown in a
Lakeland Sand. Agron. J. 73, 647651.
Marufu, L., Karanja, N., Ryder, M., 1995. Legume inoculant
production and use in east and southern Africa. Soil Biol.
Biochem. 27, 735738.
McDermott, T.R., Graham, P.H., 1989. Bradyrhizobium japonicum
inoculant mobility, nodule occupancy, and acetylene reduction
in the soybean root system. Appl. Environ. Microbiol. 55,
24932498.
Meade, J., Higgins, P., O'Gara, F., 1985. Production and storage of
Rhizobium leguminosarum cell concentrates for use as
inoculants. J. Appl. Bacteriol. 58, 517524.
Muldoon, J.F., Hume, D.J., Beversdorf, W.D., 1980. Effects of seed
and soil-applied Rhizobium japonicum inoculants on soybeans
in Ontario. Can. J. Plant Sci. 60, 399406.
Osa-Aana, L.O., Alexander, M., 1982. Clays and the survival of
Rhizobium in soil during dessication. Soil Sci. Soc. Amer. J. 46,
285288.
Paczkowski, M.W., Berryhill, D.L., 1979. Survival of Rhizobium
phaseoli in coal-based legume inoculants. J. Appl. Environ.
Microbiol. 38, 612615.
Provorov, N.A., Saimnazarov, U.B., Tanriverdiev, T.A., Simarov,
B.V., 1994. The contributions of plant and bacteria genotypes in
the growth and nitrogen accumulation of inoculated alfalfa.
Plant Soil 164, 213219.
Rennie, R.J., Kemp, G.A., 1983a. N
2
xation in eld beans
quantied by
15
N isotope dilution. I. Effect of strains of
Rhizobium phaseoli. Agron. J. 75, 640644.
Rennie, R.J., Kemp, G.A., 1983b. N
2
xation in eld beans
quantied by
15
N isotope dilution. I. Effect of cultivars of
beans. Agron. J. 75, 645649.
Rice, W.A., Olsen, P.E., 1992. Effects of inoculation method and
size of Rhizobium meliloti population in the soil on nodulation
of alfalfa. Can. J. Soil Sci. 72, 5767.
Roughley, R.J., Vincent, J.M., 1967. Growth and survival of
Rhizobium spp. in peat culture. J. Appl. Bacteriol. 30, 362376.
Sattar, M.A., Quader, M.A., Danso, S.K.A., 1995. Nodulation, N
2
xation and yield of chickpea as inuenced by host cultivar and
Bradyrhizobium strain differences. Soil Biol. Biochem. 27,
725727.
Semu, E., Hume, D.J., 1979. Inuence of soybean inoculation and
nitrogen levels on populations and serogroups of Rhizobium
japonicum in Ontario. Can. J. Microbiol. 25, 739745.
Shishido, M., Pepper, I.L., 1990. Identication of dominant
indigenous Rhizobium meliloti by plasmid proles and intrinsic
antibiotic resistance. Soil Biol. Biochem. 22, 1116.
Skinner, F.A., Roughley, R.J., Chandler, M.R., 1977. Effect of yeast
extract concentration on viability and cell distortion in
Rhizobium spp. J. Appl. Bacteriol. 43, 287297.
Smith, R.S., 1992. Legume inoculant formulation and application.
Can. J. Microbiol. 38, 485492.
Somasegaran, P., Bohlool, B.B., 1990. Single-strain versus multi-
strain inoculation: effect of soil mineral N availability on
rhizobial strain effectiveness and competition for nodulation on
chick-pea, soybean and dry bean. Appl. Environ. Microbiol. 56,
32983303.
J.H.G. Stephens, H.M. Rask / Field Crops Research 65 (2000) 249258 257
Sparrow Jr., S.D., Ham, G.E., 1983. Survival of Rhizobium phaseoli
in six carrier materials. Agron. J. 75, 181184.
Sprent, J.I., Stephens, J.H., Rupela, O.P., 1986. Environmental
effects on seasonal N
2
-xation. In: Summereld (Ed.),
Abstracts of the International Food Legume Research Con-
ference on Pea, Lentil, Fababean and Chickpea. Matinus
Nijhoff, Dordrecht, p. 47.
Thompson, J.A., 1980. Production and quality control of legume
inoculants. In: Bergerson, F.S. (Ed.), Methods for Evaluating
Biological Nitrogen Fixation. Wiley, New York, pp. 489533.
van Rensburg, H.J., Strijdom, B.W., 1982. Competitive abilities of
Rhizobium meliloti strains considered to have potential as
inoculants. Appl. Environ. Microbiol. 44, 98106.
Winaro, R., Lie, T.A., 1979. Competition between Rhizobium
strains in nodule formation: interaction between nodulation and
non-nodulating strains. Plant Soil 51, 135142.
258 J.H.G. Stephens, H.M. Rask / Field Crops Research 65 (2000) 249258