Features Inoculants Seed & Chemical
What makes a better inoculant?

Two German scientists, Hellriegel and Wilfarth, discovered the symbiotic relationship
between rhizobia bacteria and legumes in 1886. Subsequently, in 1890, two other
German scientists, Nobbe and Hiltner, showed the advantage of inoculating legumes
with rhizobia. This soon led to the start of commercial inoculants in legume
production. So, do inoculant companies really keep on 'discovering' new improved
strains – or is that just a marketing gimmick?

Inoculants can produce well-nodulated roots.

Dr. Kevin Vessey, dean of graduate studies at Saint Mary's University at Halifax,
Nova Scotia, says that indeed, inoculant manufacturers are continually discovering
new and better strains of inoculants for better performance in the field: it
is not a marketing gimmick at all. He says that while only certain species of
rhizobia will infect and fix nitrogen in certain species of legumes, different
strains of a specific rhizobia species do infect and fix more nitrogen than
other strains in the same crop. "The tough choice is to select one or two
strains from the millions of different strains of rhizobia that could be found
in the world to go into an inoculant," explains Vessey.

How do inoculants work?

The process is relative complex, and quite remarkable, as Mother Nature tends
to be. Infection of the root hair by rhizobia bacteria is the first step in
nitrogen fixation for most Canadian crops (in some crops like peanut, the infection
process is different).

The soil bacteria, rhizobia, identify a suitable plant host by the chemicals
that are released by the plant roots. Like dating, the plant root and bacteria
send co-ordinated chemical signals back and forth. A localized enzyme produced
by the root causes wall softening. This chemical 'wink' allows the root hair
to curl around the bacteria, trapping the rhizobia.

Research has shown that within one minute of adding rhizobia to a susceptible
host, attachment of rhizobia to root hairs can be observed. Root hair curling
generally begins within three to five hours. The first infection threads are
generally visible in three days, and the first nodules may be visible within
six to 12 days. Nitrogen fixation may begin within 15 days of initial infection.

Another fascinating part of nitrogen fixation is the symbiotic relationship
between bacteria and plant host. The plant provides a suitable environment for
the bacteria to live, complete with a supply of food and carefully regulated
environment. The bacteria co-operate by producing leghaemoglobin (legume haemoglobin)
inside the nodule, which binds with oxygen. Nitrogen fixation cannot function
in the presence of oxygen, so the production of leghaemoglobin is critical to
the process. Noteworthy: leghaemoglobin is red, which is why the inside of a
healthy, nitrogen-fixing nodule is pink.

Species versus strain

Originally, scientists were only concerned about the species of bacteria that
would infect a host plant. For example, Rhizobium leguminosarum will
infect peas, lentils and chickling vetch, while Rhizobium phaseoli will
infect dry beans, and Bradyrhizobium japonicum will infect soybeans.
There are currently 38 known species of root and stem nodulating bacteria, but
the number is growing. More recently, strain selection has become important
in optimizing N fixation.

To better understand the difference between strain and species, think of species
as a crop, while the strain is a crop variety. When put in that context, understanding
the difference in strain performance can be more easily appreciated.

Mary Leggett, research team leader on strain research with Philom Bios at Saskatoon,
Saskatchewan, says the search for higher performing strains focusses on several
objectives. The strain must fix more nitrogen than other known strains. The
strain must be compatible with the plant host as well, since the strain can
be too aggressive and become almost parasitic on the plant. The strain also
must be able to live and compete in the soil in the area immediately around
the germinating seed.

"It's like a war zone in the soil with all these different types of micro-organisms
competing for nutrients in the rhizosphere, the microscopic area immediately
around the root," explains Leggett. "We try to find the most effective
strain, which is the most competitive. In that way, we can be assured that the
strain will provide better nitrogen fixation."

Vessey classifies these objectives into 'infective' and 'effective'. Infective
characteristics of a strain mean that the bacteria will grow well in the soil
and out-compete other bacteria to infect the plant root hairs, including less
infective strains of the same rhizobia bacteria. Effective means the strain,
once inside the plant's nodules, is very good at fixing nitrogen.

High performing strains should also be genetically stable, tolerant of various
environmental conditions in the soil (including pH, and environmental conditions),
and be easy and economical to grow in the manufacturing facility.

Discovery takes patience

So where do inoculant companies find these strains? In a grower's backyard is
one of the answers. Leggett explains that researchers literally dig up samples
from local fields, pastures and native grasslands looking for new promising
strains. The rhizobia bacteria are isolated, identified and tested for performance
in the laboratory as the first screen.

"We collect hundreds of samples a year and we can isolate and identify
200 or 300 isolates for screening from these samples," says Leggett. To
help focus the processing of the samples, bacteria are grown on media that will
only allow the rhizobia species of interest to grow. A single sample contains
millions of micro-organisms, but the vast majority are not of interest. "It
is a time consuming process and takes a significant amount of resources, but
the method does yield results."

The rhizobia strains in commercial inoculants in Canada are not genetically
modified, thus natural selection is used. "The value of a 'shot gun' approach
of searching through hundreds of plant and soil samples is that it draws from
a large genetic pool of micro-organisms," explains Vessey. As a result,
there is always the potential that a better performing strain of rhizobia is
still out there.

Hi-tech genetic tests are used in strain identification to help ensure researchers
are not chasing the known 'dogs' of the strain world. In peas and lentils, researchers
can use a 'plasmid' test to search for small bits of DNA that help in strain
identification. Another genetic test is the 'AFLP' test, where the DNA is basically
mashed up and sections of enzymes are examined.

Biological profiles also can be used to identify strains. For example, one
test looks at the carbohydrate profile that a strain uses during respiration.

Testing. Testing. Testing

After promising strains are identified in the laboratory, researchers take them
to the greenhouse to see how they compare to existing commercial inoculants.
If these trials are promising, the next step is several years of field testing
of the strain and formulations, as the carriers used to formulate the product
also undergo the same rigorous testing. For example, granular formulations consisting
of a base of clay, gypsum or peat granules include polymers, water and other
ingredients in various combinations. These formulations must be tested to ensure
they are compatible with the new strain and are capable of supporting a large
population of the selected rhizobia strain.

Similarly, the new strains and formulations must be tested for compatibility
with other seed treatments like fungicides or insecticides.

At the same time field and formulation testing is being conducted, Leggett
says the most promising strains are turned over to the manufacturing group at
Philom Bios, to determine the most effective way to multiply the strain up to
commercial quantities.

"The goal of a new commercial inoculant is to get the most infective and
effective strain into the soil, have it out-compete other bacteria and fix a
high level of nitrogen for the crop," explains Leggett. "It's not
an easy task, but we have been able to continually raise the standard of our
inoculants' value and performance."

Vessey also says that as crop varieties change, the effectiveness of strains
may also change, requiring on-going research and development. "The search
for 'new and improved' strains of rhizobia is an ongoing job for inoculant companies."
-30-