REGULAR ARTICLE
Field response of rice paddy crop to Azospirillum
inoculation: physiology of rhizosphere bacterial communities
and the genetic diversity of endophytic bacteria in different
parts of the plants
Inés E. García de Salamone & Luciana P. Di Salvo & Jhovana S. Escobar Ortega &
Paulo M. F. Boa Sorte & Segundo Urquiaga & Kátia R. S. Teixeira
Received: 12 May 2009 /Accepted: 1 July 2010 /Published online: 18 August 2010
# Springer Science+Business Media B.V. 2010
Abstract The response of rice plants to the application
of inoculant containing two Azospirillum brasilense
strains was studied under field conditions. The experi-
ment was performed as three treatments with four
replicates in randomized complete blocks arranged as
plots of 60 m2 in an area on a Vertic Argiudol soil type
in the province of Entre Ríos, Argentina. The bacterial
rhizosphere community and also the diazotrophic isolates
obtained from control and inoculated rice plants were
analyzed in relation to their physiology and biological
nitrogen fixation (BNF). The MPN of diazotrophs in the
rhizosphere varied during the ontogenic cycle. The
patterns of distribution of the microbial physiological
activities obtained by principal component analysis of
community-level physiological profiles (CLPP) showed
differences in the utilization of carbon sources by the
rhizosphere communities among treatments. Although
the analyses of DGGE 16S and nifH profiles have not
indicated that the inoculation influenced the genetic
diversity of bacterial communities among treatments,
they revealed that the banding profiles were altered in
different parts of the rice plant by each Azospirillum
inoculation treatment. These observations suggest that
physiological responses of plant tissues to the inocu-
lation may have occurred. According to agronomic
parameters of each treatment, the Azospirillum inocu-
lation increased aerial biomass at the tillering and
grain-filling stages. Although the N content accumu-
lated in rice plants increased by 16 and 50 kg ha−1, the
BNF contribution could not be estimated under our
experimental conditions by the 15N balance technique.
Based on this field inoculation experiment to rice
plants, it is noteworthy that our data suggest that due to
Azospirillum inoculation the increase of total N
accumulated in rice plants could be a tool to help
farmers to improve production and maintain high input
of plant residues, providing more organic matter to the
soil and guaranteeing sustainability of the system.
Plant Soil (2010) 336:351–362
DOI 10.1007/s11104-010-0487-y
Responsible Editor: Euan K. James.
Electronic supplementary material The online version of this
article (doi:10.1007/s11104-010-0487-y) contains
supplementary material, which is available to authorized users.
I. E. García de Salamone (*) : L. P. Di Salvo :
J. S. Escobar Ortega
Agricultural Microbiolgy Unit, FAUBA,
San Martín Ave. 4453,
Buenos Aires 1417, Argentina
e-mail: igarcia@agro.uba.ar
e-mail: ines.e.garcía@gmail.com
P. M. F. Boa Sorte : S. Urquiaga : K. R. S. Teixeira
Embrapa Agrobiologia,
BR465 Km7,
Seropédica, RJ CEP 23890-000, Brazil
P. M. F. Boa Sorte
Department of Agronomy - Soil Science,
Universidade Federal Rural do Rio de Janeiro (UFRRJ),
Seropédica, Rio de Janeiro, Brazil

Keywords Genetic diversity . Endophytic bacterial
community . Inoculant . N content . PGPB . Rice
Introduction
Rice (Oryza sativa L.) is the staple food for a great
proportion of the world’s population. The northeast
region of Argentina produces high yields of this
cereal, but it is important to search for sustainable
strategies to increase and maintain high levels of
production. Current approaches involve application of
agro-chemicals (fertilizers, pesticides and herbicides)
that, in general, increase costs and can generate
negative environmental impacts.
Nitrogen (N) is one of the most important elements
in plant nutrition, particularly in Latin American
countries where most soils show low N availability
and the agricultural subsidies to N fertilizer are low or
nonexistent. For these reasons, there is a great interest
in exploring the diversity of plant growth-promoting
bacteria (PGPB) as substitutes for some chemical
inputs to agriculture. N2-fixing microorganisms, or
diazotrophs, can be inoculated into crops to increase
the number of beneficial organisms contributing to
plant growth and crop production. In general, it has
been observed that cereal plants are capable of
associating with diverse bacterial species including
diazotrophs that can promote growth (Okon and
Labandera-Gonzalez 1994; García de Salamone et
al. 1996; Rodrigues et al. 2008). Most of these PGPB
can colonize the exterior of plant roots and aerial
tissues in several plants, and can also associate
endophytically to most of them, including rice
(Baldani and Baldani 2005; Döbereiner and Pedrosa
1987; Olivares et al. 1996; Rodrigues et al. 2008).
The inoculation of cereal crops with PGPB is
relevant for both economic and ecological reasons. In
Argentina and several other countries, Azospirillum
strains have been used as PGPB in inoculation field
trials, mostly because of their ubiquitous behavior and
association with grasses and cereals, such as rice,
wheat and maize. Azospirillum spp. have shown great
versatility to adapt to diverse soil conditions and have
been isolated from the rhizosphere, the root surface and
the inside of several tissues of wide variety of crops
and wild plants worldwide (Bashan et al. 1991, 2004;
Döbereiner and Pedrosa 1987). This genus belongs to
the α subclass of the proteobacteria, and very recently
several new species have been described. Nonetheless,
A. brasilense is the species that has been widely used
for both experimental and commercial formulations.
Plant–bacteria associations can occur on different
levels of interaction. Thus, the genetic characteristics of
both bacteria and host plant can define the level of the
inoculation response during association (García de
Salamone and Döbereiner 1996; Olivares et al. 1996;
Azevedo et al. 2005). When the same strain is applied as
inoculant to different crops, significantly different levels
of response can be obtained, and several mechanisms
of plant growth promotion have been attributed to
them (Bashan and Holguin 1997; Bashan et al. 2004).
Under controlled conditions, when Azospirillum
amazonense strains were used as inoculants, it has been
shown that contribution of the biological nitrogen fixation
(BNF) for rice productivity could range from 9.2 to
27.7% of Ndfa accumulation in the plant improving the
potential of BNF in the inoculated plants up to 4-fold
relative to noninoculated controls (Rodrigues et al.
2008). Using selected strains of diazotrophs which can
supply high levels of N through BNF could further
increase the efficiency of this metabolic activity under
field conditions. In this regard, previous data demon-
strated that BNF could represent a significant N supply
for the production of two commercial genotypes of
maize that were inoculated with selected A. brasilense
strains (García de Salamone et al. 1996).
The objective of this work was to study, under field
conditions, the eco-physiological response of rice
plants to the inoculation with two A. brasilense strains
previously characterized by their ability to fix N2 in
association with maize plants (García de Salamone et al.
1996). The rhizosphere bacterial community of inoc-
ulated rice plants and isolates of diazotrophic bacteria
were analyzed in relation to BNF and their physiology.
In addition, the genetic diversity of the endophytic
bacterial communities, including diazotrophs, was
characterized in different parts of rice plants.
Materials and methods
Bacterial strains and inoculant preparation
Azospirillum brasilense strains 40 M (GenBank
accession number HM002661) and 42 M (GenBank
accession number HM002662), isolated from inside
the roots of field-grown maize (García de Salamone
352 Plant Soil (2010) 336:351–362