Survey of indigenous diazotrophic
bacteria
associated to maize in Uruguay
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Summary Diazotrophs have been cultured from both
the plant interior and root environment of maize. Studies using stable
isotope (15N) incorporation imply that bacteria in association with maize are
actively fixing nitrogen. This
evidence suggests that diazotroph-maize associations have potential benefits
to agriculture if the yields of maize can be sustained in the absence of high
N fertilizer inputs. As nitrogen
fertilization is an important cost in maize production and environmental
protection, the enhancement of maize-diazotroph association would be of great
economic value to Uruguay. Data in
support of plant growth benefits to maize accrued via nitrogenase activity
are lacking in this country.
Investigations on diazotroph-maize associations, such as plant
genotype and cultivation conditions, and bacterial community structure and
population dynamics of associating diazotrophs should illuminate those
interactions that can be further explored and manipulated to enhance
biological nitrogen fixation and sustainable production of maize plants. This
study aims to enhance biological nitrogen fixation in maize in Uruguay. To this end, isolation, characterization
and quantification of nitrogen fixed of the predominant endophytic
diazotrophs associated to various maize varieties will be performed. Scientific background Breeding
programs for crop plants are designed to improve agronomic characteristics
such as yield, fertilizer use efficiency and disease resistance. These programs do not typically consider
interactions between plants and soil microbial communities like diazotrophic
bacteria. It is well known that maize
plants can establish natural associations with various N2-fixing bacteria
like Azospirillum[i],
Klebsiella[ii]
[iii],
Pantoea, Herbaspirillum and Bacillus[iv][v]. All or some of these bacteria may be
responsible for supplying the plants with fixed N2. The potential of associative
diazotrophic bacteria to promote the growth of many cereals and grasses has
been demonstrated[vi]
by several authors. In addition to N2 fixation, associative diazotrophs exert
their positive effects on plant growth directly or indirectly through a
combination of different mechanisms.
Diazotrophs can affect plant growth directly by the synthesis of
phytohormones and vitamins, inhibition of plant ethylene synthesis, improved
nutrient uptake, enhanced stress resistance, solubilization of inorganic
phosphate and mineralization of organic phosphate. Indirectly, diazotrophs are able to decrease or prevent the
deleterious effects of pathogenic microorganisms, mostly through the
synthesis of antibiotics and/or fungicidal compounds, through competition for
nutrients or by the induction of systemic resistance to pathogens. In addition, they can affect the plant
indirectly by interacting with other beneficial microorganisms. This suggests that there is potential for
a plant growth benefit from diazotrophic bacteria if the appropriate bacteria
and/or plant genotype and growth conditions are met. In view of the above, this study aimed to
identify indigenous diazotrophic bacteria associated to maize varieties in
Uruguay with increased plant productivity and in particular, nitrogen
fixation ability. Scientific scope of the project Nitrogen
is an essential plant nutrient. It is the nutrient that is most commonly
deficient in soils, contributing to reduced agricultural yields throughout
the world. Nitrogen can be supplied
to crops by biological nitrogen fixation (BNF), a process which is becoming
more important not only for reducing energy costs, but also in seeking more
sustainable agricultural production.
Therefore, nitrogen-fixing microorganisms could be important
components of sustainable agricultural systems. Maize is an important food crop in Uruguay and Latin
America. The screening of plant
genotype for higher contributions of BNF has been cited to be the key to the
replacement of N fertilizers in several important crops like sugar cane,
rice, wheat, maize and others. This
study aims to enhance biological nitrogen fixation in maize in Uruguay. To this end, isolation, characterization
and quantification of nitrogen fixed of the predominant endophytic
diazotrophs associated to various maize varieties will be performed. Detailed work plan for first year, including proposed
methods and techniques Most
reported isolations of maize endophytes result from work done with plants
cultivated in the field. In many
cases, the working hypothesis is that the inoculum source of endophytic
diazotrophs is the soil from which the crop is frequently cultivated. The Rhizobium-legume model follow this
pattern but not is the case of sugar cane, where the endophyte A.
diazotrophicus has been found in the tissue of sugar cane, but has not been
recovered from soil between crop rows [vii] [viii]. Several studies have reported the
isolation of diazotrophs from surface sterilized seeds of grasses, including
turf grass, rice and maize7. Maize
surface sterilized seeds possessed 105-106 bacteria per gram fresh weight[ix].
Plants grown under sterile conditions from surface-sterilized seeds also
contained stem bacteria but at levels 100-fold lower than plants cultured in
non-sterile soil. This suggests that
the endophitic bacteria can be transmitted either through seed or through
soil. Inoculum seed trasmition is
potentially important in annual specie like maize and suggest a simple method
of inoculation of plant. In
this study indigenous diazotrophic bacteria able to colonize plant tissues,
associated to maize in Uruguay will be investigated. |
Step 1 : Isolation and identification of diazotrophic endophytes in maize Uruguay
has a collection of maize germplasm that we will use for this study[x][xi]..
The most common varieties of maize used in Uruguay will be used to isolate diazotrophic
endophytes from seed, stems tissue and roots previously surface
sterilized. Isolation of diazotrophic
bacteria will be done using a modified semisolid N-free LG1 medium[xii], The microorganisms of interest will be
purified and characterized by morphologic and cultural methods. Step 2 : Screening of diazotrophic endophytes and identification of strains with the best abilities to colonize, fix N, and promote plant growth. Microbial
isolates characterized previously will be screened for nitrogen fixation
effectiveness by a single-inoculation trial in soil microcosms. The parameters evaluated will be dry
biomass accumulation (root and shoot), N and P uptake and N2-fixation (15N). A further field test could be performed in
the future. Step 3: Screening of maize germplasm for low mineral N dependence and high
nitrogen fixation. An
association that contributes to the nitrogen nutrition of maize might be most
easily identified when plant are cultivated with little dependence on
exogenously supplied nitrogen. The rate of N fertilizer application can
affect not only the activity of the indigenous population of nitrogen fixers,
but also the activity of inoculated strains. Therefore a greenhouse
experiment will be conducted to study the combined effect of diazotrophic
bacteria activity and mineral nitrogen fertilisation.. Surfaced
sterilized seeds of previous selected varieties of maize will be planted in
non sterile sand-soil and watered with 3 levels of N fertilizers (low, medium
and high).
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12. Cavalcante and Döbereiner |