Field Site-Specific Effects of an Azospirillum Seed Inoculant on Key Microbial Functional Groups in the Rhizosphere

Abstract

The beneficial effects of plant growth–promoting Rhizobacteria (PGPR) entail several interaction mechanisms with the plant or with other root-associated microorganisms. These microbial functions are carried out by multiple taxa within functional groups and contribute to rhizosphere functioning. It is likely that the inoculation of additional PGPR cells will modify the ecology of these functional groups. We also hypothesized that the inoculation effects on functional groups are site specific, similarly as the PGPR phytostimulation effects themselves. To test this, we assessed in the rhizosphere of field-grown maize the effect of seed inoculation with the phytostimulatory PGPR Azospirillum lipoferum CRT1 on the size and/or diversity of selected microbial functional groups important for plant growth, using quantitative polymerase chain reaction and/or Illumina MiSeq metabarcoding. The functional groups included bacteria able to fix nitrogen (a key nutrient for plant growth), producers of 1-aminocyclopropane-1-carboxylate (ACC) deaminase (which modulate ethylene metabolism in plant and stimulate root growth), and producers of 2,4-diacetylphloroglucinol (an auxinic signal enhancing root branching). To test the hypothesis that such ecological effects were site-specific, the functional groups were monitored at three different field sites, with four sampling times over two consecutive years. Despite poor inoculant survival, inoculation enhanced maize growth. It also increased the size of functional groups in the three field sites, at the maize six-leaf and flowering stages for diazotrophs and only at flowering stage for ACC deaminase and 2,4-diacetylphloroglucinol producers. Sequencing done in the second year revealed that inoculation modified the composition of diazotrophs (and of the total bacterial community) and to a lesser extent of ACC deaminase producers. This study revealed an ecological impact that was field specific (even though a few taxa were impacted in all fields) and of unexpected magnitude with the phytostimulatory Azospirillum inoculant, when considering microbial functional groups. Further methodological developments are needed to monitor additional functional groups important for soil functioning and plant growth under optimal or stress conditions.

Effect of Inoculation Level on the Impact of the PGPR Azospirillum lipoferum CRT1 on Selected Microbial Functional Groups in the Rhizosphere of Field Maize

“…Azospirillum lipoferum CRT1 is a natural PGPR isolate that is commercially used on maize to stimulate plant growth, and field yield, with a peat-based seed-coating formulation providing 10 5-6 CFU of bacteria per seed [29,30]. Nevertheless, phytostimulation can also be achieved at lower inoculation levels, and we evidenced positive effects of A. lipoferum CRT1 on maize physiology with only 10 4-5 inoculant cells per seed [20,21,31]. In addition to direct effects on maize physiology [29], inoculation of maize seeds with A. lipoferum CRT1 affects the bacterial community of the rhizosphere [21,32]; this includes specific microbial functional groups important for plant growth [21], such as diazotrophs (nitrogen fixation), ACC deaminase producers (regulators of ethylene metabolism in roots), and producers of 2,4-diacetylphloroglucinol, a root-branching signal with auxinic effects [33].…”

“…Nevertheless, phytostimulation can also be achieved at lower inoculation levels, and we evidenced positive effects of A. lipoferum CRT1 on maize physiology with only 10 4-5 inoculant cells per seed [20,21,31]. In addition to direct effects on maize physiology [29], inoculation of maize seeds with A. lipoferum CRT1 affects the bacterial community of the rhizosphere [21,32]; this includes specific microbial functional groups important for plant growth [21], such as diazotrophs (nitrogen fixation), ACC deaminase producers (regulators of ethylene metabolism in roots), and producers of 2,4-diacetylphloroglucinol, a root-branching signal with auxinic effects [33]. Therefore, it is likely that phytostimulation by this PGPR involves a combination of direct effects on the plants, as well as indirect effects on the indigenous rhizosphere microbial community.…”

“…Effect of inoculum level of Azospirillum lipoferum CRT1 on root and shoot parameters of maize grown at the three fields sites L (Chatonnay), FC (Sérézin-la-Tour) and C (Saint Savin). Formulation F1 corresponds to the peat matrix used to coat seeds with low inoculant concentration, and the corresponding data in this table originate from [21]. In formulation F2, A. lipoferum CRT1 was used at higher inoculant concentration.…”

“…In the context of ecological awareness in agronomy, biofertilisation with microorganisms constitutes an attractive alternative to chemical inputs, which are polluting and decrease soil fertility [13][14][15][16]. Various studies reported that PGPR inoculant functioning under field conditions is impacted by soil type and plant genotype, as well as the PGPR strains used [2,9,10,[17][18][19][20][21].…”

“…The objective of this work was to assess the effect of a reduced inoculum level (10 4-5 cells.seed −1 ; formulation F1) of A. lipoferum CRT1 on its host physiology and rhizosphere microbial communities, in comparison with a standard inoculum level (10 5-6 cells.seed −1 ; formulation F2) under agronomic conditions. These maize inoculation experiments were carried out in parallel at three different field sites; additionally, results obtained with the F1 formulation on maize photosynthesis efficiency, metabolome, growth and yield have been previously reported [20], along with findings on nitrifying and denitrifying communities in bulk soil [31], as well as diazotroph communities, ACC deaminase producer communities and 2,4-diacetylphloroglucinol producer communities in the rhizosphere [21]. However, formulation F1 data-enabling to compare the two inoculation levels has not been reported, and this was the focus of this study.…”