Download Identification of exogenous growth stimulants or N

AR0904 Executive Summary
Identification of exogenous growth stimulants or nitrogen fixation from
A.caulinodans.
Azorhizobium caulinodans is a tropical, symbiotic N-fixing bacterium that has
previously, yet only tentatively, been identified as being capable of forming a
beneficial relationship with Wheat. Previous studies by ADAS/University of
Notting ham (CE0153) indicated that inoculation of wheat with A.caulinodans
may offer the potential to supply agriculturally useful quantities of nitrogen to the
cropping system as well as providing hormonal stimulation to root growth, and
therefore enhanced uptake of nitrogen and perhaps other nutrients. The nature
of these hormonal stimulants was not confirmed, biological N fixation was not
demonstrated categorically and the location of the N-fixation event (is it truly
endophytic symbiosis or free-living N-fixation?) was not confirmed. The work
reported here elucidates all three areas. The fundamental mode of action of the
relationship is described. The work supports DEFRA objectives by seeking to
improve the sustainability of cereal production systems by reducing reliance on
inorganic N fertilisers.
In brief, the work addressed the following objectives:
1. To identify the nature of the non N-fixation component of growth stimulation in
inoculated plants.
2. To establish the magnitude of the N-fixation component of growth stimulation
through 15N assay.
3. To establish the site of N-fixation.
4. To establish the longevity of A.caulinodans in non-sterile soil.
For each objective, experiments were set up which used the same basic sent of
treatments. Plants were grown under controlled environments; the day/night
temperatures were set to 18/8oC, with a 12 hour photoperiod (800 ∏m m-2 s-1) to
simulate late spring conditions in the UK. In all experiments the soil was a sandy
loam supplied form ADAS Gleadthorpe, with a residual N content of 11mg kg –1.
In order to investigate whether biological nitrogen fixation (BNF) was taking
place, wild type A.caulinodans (nif+) was compared with the non N-fixing mutants
nif-. In order to identify the nature of the non N-fixation component of growth
stimulation, inoculated plants received either drenches of nif- or nif+, the
supernatant from nif- and nif+, or untreated controls applied to plants. The indol
acetic acid (IAA) content of all suspensions was measured. It was hypothesised
that application of the nif- or nif+ supernatant would show a similar growth
enhancement if the bacteria were producing IAA. Inoculation with both nif+
A.caulinodans and nif+ supernatant significantly increased total dry weight of
plants, as did inoculation with nif- or nif+ supernatant. There were no significant
differences in the N content of the bacterial cultures or their supernatants. IAA
was identified in all bacterial cultures and supernatants. HPLC and a colormetric
test were carried out to detect whether the bacteria were producing IAA in
culture. These data suggested that wheat growth enhancement was due to
hormonal stimulation rather than N fertiliser effects. Results from 15N studies
confirmed the absence of BNF. There was no significant difference in the 15N
dilution of plants from different treatments showing that BNF is unlikely to be
increasing plant growth. There was an increase in early plant growth observed,
consistent with an effect of plant growth substances on seedling growth.
Experiments undertaken to identify the site of bacterial colonisation examined for
the long-term presence of bacteria within plants, on the surface of plants or in the
soil. A.caulinodans were found in the soil of inoculated plants 21 days after
sowing (DAS; the drench having been applied 15 DAS). A.caulinodans were
isolated from the root surface between 28 and 56 DAS and there were no
differences in the population sizes of either nif- or nif+ bacteria. Bacterial
numbers declined from 28 to 56 DAS and by 77 DAS there were no live bacteria
on the root surface. Possible causes of this lack of longevity were that the
bacteria could not survive under temperate conditions, the wheat root exudates
may not have provided all necessary nutrients or that the bacteria could not find
an empty niche.
To summarise, these experiments not only provide evidence that the plants are
not benefiting from N-fixation but they also provide strong evidence for plant
growth enhancement due to the production of plant growth substances by the
bacteria in culture. It also appears that the differences in the effect on plant of
inoculation with the nif- and nif+ bacteria are due to differences in the production
of IAA by the bacteria in culture. However, it should be remembered that
diazotrophic bacteria have been shown to produce plant growth substances other
than IAA in culture, which have also been shown to increase plant growth.
These results indicate that the wild type (nif+) and mutant (nif-) A.caulinodans
provide a small plant growth boost through hormone production, they have no
future credible role in replacing the need for N fertiliser in agricultural systems,
however the potential for increasing fertiliser recovery by the application of plant
growth substances may be worthy of further investigation.