212 M. Lin et al. Applied Soil Ecology 15 2000 211–225
communities remained, with a few, varying, exceptions, largely unchanged in spite of the release of strain A1501R. However, significant differences in the utilization of selected substrates were observed between the control and inoculated soils, as
evidenced by the application of PCR–DGGE to the bacterial communities inhabiting selected wells of the Biolog plates. In particular, strain A1501R was found to be highly competitive in the presence of lactic acid. © 2000 Elsevier Science B.V. All
rights reserved.
Keywords: Alcaligenes faecalis ; Survival rice rhizosphere; PCR–DGGE Biolog
1. Introduction
The development and agricultural application of bacterial inoculants, either unmodified or genetically
modified to improve their function, has attracted con- siderable interest in the last decade You and Zhou,
1989, 1991; van Elsas et al., 1991; De Leij et al., 1995; van Overbeek et al., 1997; van Veen et al.,
1997. An important area has been the application of associative diazotrophic bacteria to crops growing
in soils with nitrogen deficiencies. In particular, in- tensive rice cropping regimes such as those common
in Chinese agriculture have been indicated to benefit from such associations You and Zhou, 1989, 1991;
You et al., 1991; Ueda et al., 1995.
The diazotrophic bacterium Alcaligenes faecalis A1501, originally obtained from paddy soil in China
You and Zhou, 1989; You et al., 1995, has been shown to be a key organism for application to rice
crops, either in inoculant mixtures or as a single in- oculant You and Zhou, 1991; You et al., 1991. This
organism has been successfully used in experimental inoculant mixtures in the open field Lin, 1997. To
enhance the in situ inoculant nitrogen fixation rates, genetically modified GM derivatives have been pro-
duced for strain 1501, as well as for other components of inoculant mixtures, i.e. nitrogen-fixing strains of
Enterobacter cloacae
and Klebsiella oxytoca Fu- jii et al., 1991; You and Zhou, 1991, 1989; You et
al., 1995. The modifications in these strains were brought about via the introduction of an exogenous,
constitutively expressed, nifA regulatory gene on a plasmid or inserted into the chromosome You and
Zhou, 1989, 1991. The resulting GM strains thus displayed an enhanced capacity to fix nitrogen and
even fixed nitrogen under high-ammonia conditions You et al., 1995.
Field experiments with rice and soybean inoculated with the GM A. faecalis A1501 inoculant in mixes
have been performed in south China since 1989 You et al., 1995. The main purpose of these field releases
has been the improvement of crop productivity and economization of nitrogen fertilizer, as it is known that
intensive rice cropping can cause a depletion of avail- able nitrogen Reichardt et al., 1997. In rice, nitro-
gen fixation by the modified strain was about 15–20 raised over unmodified controls, as evidenced by using
the
15
N isotope dilution technique Lin, pers. comm.. Yield increases of field-grown rice were about 5–12
when GM strains were used and 3–7 with parent strains You et al., 1995. The A. faecalis inoculant
strain has further been proposed and tested for use in rice and soybean cropping in salt-affected or degraded
soils, due to its beneficial characteristics, such as the production of the plant growth hormone indole acetic
acid and tolerance to salt Lin et al., 1992.
To understand the performance and putative adverse effects of the A. faecalis inoculant in soil, monitoring
of its fate and impact over time is needed. Such an as- sessment is particularly crucial as a prelude to future
commercial large-scale releases. In addition to data on inoculant persistence and potential spread, its ef-
fect on indigenous microbial communities should be understood, for instance via monitoring of changes in
the diversity and activity of microbial communities in soil Wunsche et al., 1995; van Elsas et al., 1998.
As outlined recently, a polyphasic approach to fate and impact assessments, encompassing the use of
combined traditional and advanced molecular tech- niques, is recommended van Elsas et al., 1998.
Among the molecular techniques, PCR-based meth- ods that use soil DNA are very suitable for the assess-
ment of inoculant fate Smalla et al., 1993 as well as the indigenous microbial community structure.
Using conserved bacterial primers, ribosomal RNA gene based PCR of soil DNA followed by DGGE
PCR–DGGE Myers et al., 1987; Muyzer et al., 1996, can generate molecular profiles that describe
M. Lin et al. Applied Soil Ecology 15 2000 211–225 213
the dominant members of soil microbial communities Heuer and Smalla, 1997b; Duineveld et al., 1998.
Furthermore, the metabolic potential of microbial communities can be assessed via community level
physiological profiling CLPP using the Biolog GN microtiter plate setup Garland and Mills, 1991; Gar-
land, 1997. In spite of its obvious drawbacks Smalla et al., 1998, this method has been widely used as
a rapid and powerful community-level approach to study the potential activity of microbial populations
in natural environments Winding, 1994; Zak et al., 1994; Garland, 1997; Heuer and Smalla, 1997b; Hitzl
et al., 1997; Insam, 1997; Knight et al., 1997. Combi- nation of the two methods thus allows a simultaneous
functional and structural assessment of the impact of the release of inoculant strains into soils Akkermans
et al., 1995; Smalla et al., 1998.
This study aimed to assess the fate and impact of A. faecalis strain A1501R in soil cropped with
rice in microcosms. To achieve this objective, the autecology of the inoculant strain was monitored by
different methods. Furthermore, the impact of the A. faecalis
strain on the soil and rhizosphere microbiota was assessed by comparing the PCR–DGGE profiles
and Biolog GN metabolic fingerprints of microbial communities between soil samples inoculated or not
with strain A1501R.
2. Materials and methods
