Several specific primers for the
nifH gene were tested with different pure telluric N
2-fixing strains. A PolF/PolR primer set provided successful amplification of 19 representative N
2-fixing ...strains. Three restriction enzymes,
HaeIII,
NdeII and
MnlI, chosen for restriction fragment length polymorphism (RFLP) analyses, were the most discriminating for the study of
nifH gene diversity as they resulted in differences between strains at the species level. Amplification by selected primers and RFLP were applied to assess the genetic diversity of the
nifH gene pool in soil. Pair soils, one under cultivation, the second under permanent pasture, were found to harbor a contrasting diversity of
nifH genes. Pure strain profiles could not be recognized in the
nifH soil patterns. Using the simple procedure described, it was shown that the structure of nitrogen fixers in soil was influenced by soil functioning.
Azospirillum lipoferum is a soil bacterium known for its ability to colonize roots and to promote plant growth. Recently, a laccase-like polyphenol oxidase activity has been reported in a strain ...isolated from the rhizosphere of rice. Purification to apparent homogeneity of
A. lipoferum laccase was achieved after cell disruption by sonication, protein solubilization by Triton X-100, acetone and protamine precipitation, hydroxyapatite column chromatography in the presence of urea (5 M) and NaCl (2 M), and finally by conventional hydroxyapatite column chromatography. After this final step, laccase was in an inactive form but could be reactivated by adding sodium dodecyl sulfate (SDS). Based on gel electrophoresis results, we suggest that native
A. lipoferum laccase is composed of a catalytic polypeptide chain with low molecular mass (16.3 kDa) and one or two regulatory/structural chains with high molecular mass (81.5 kDa). The purified enzyme was found to be thermostable up to 70°C for 10 min, had an optimal pH of 6.0, and was inhibited by tropolone, a known inhibitor of metal-containing enzymes. Although the natural substrate of this enzyme is unknown, its biochemical characterization may facilitate further investigations on the ecological role of this laccase in the process of root colonization by
A. lipoferum.
Screening of 10 environmental samples (mainly of rhizospheric origin) for lytic activity against two bacterial phytopathogens,
Pseudomonas syringae
pv.
tomato
DC3000 (CFBP2212) and
Xanthomonas ...hortorum
pv.
vitians
(CFBP3979), revealed that four samples harboured phages that were active against one strain. Only one sample, composed of an artisanal nettle liquid manure, contained phages able to lyse both strains. Electron microscopy revealed the presence of tailed bacteriophages, with all phages isolated on the
Xanthomonas
strain displaying a contractile tail typical of members of the family
Myoviridae
, whereas phages isolated on the
Pseudomonas
strain were related to members of the family
Siphoviridae
and short-tailed members of the family
Podoviridae.
Sequence analysis of the two
Podoviridae
-like bacteriophages isolated on
Pseudomonas syringae
pv.
tomato
, Pst_GM1 isolated from nettle manure and Pst_GIL1 isolated from infected lettuce leaves, revealed (i) strong homology between the two isolated phages, (ii) a high degree of sequence similarity to various phages isolated from various environments and from different geographical locations, and (iii) similarity of these phages to members of the family
Autographiviridae
, and more precisely, the genus
Ghunavirus
. Further investigation of the potential of nettle manure to host phages that could be active against a wider range of strains revealed that it contained phages active against 10 phytopathogens (out of 16 tested). Thus, nettle manure (and likely other plant manures) could represent a valuable source of phages, especially those targeting bacterial phytopathogens, in the same way that anthropized environments such as sewage are widely used as sources of phages active against opportunistic or acute pathogens of humans.
Most Azospirillum plant growth-promoting rhizobacteria (PGPR) benefit plant growth through source effects related to free nitrogen fixation and/or phytohormone production, but little is known about ...their potential effects on plant physiology. These effects were assessed by comparing the early impacts of three Azospirillum inoculant strains on secondary metabolite profiles of two different maize (Zea mays) cultivars. After 10 d of growth in nonsterile soil, maize methanolic extracts were analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC) and secondary metabolites identified by liquid chromatography/mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR). Seed inoculation resulted in increased shoot biomass (and also root biomass with one strain) of hybrid PR37Y15 but had no stimulatory effect on hybrid DK315. In parallel, Azospirillum inoculation led to major qualitative and quantitative modifications of the contents of secondary metabolites, especially benzoxazinoids, in the maize plants. These modifications depended on the PGPR strain × plant cultivar combination. Thus, Azospirillum inoculation resulted in early, strain-dependent modifications in the biosynthetic pathways of benzoxazine derivatives in maize in compatible interactions. This is the first study documenting a PGPR effect on plant secondary metabolite profiles, and suggests the establishment of complex interactions between Azospirillum PGPR and maize.
Background and aims Many plant-beneficial microorganisms can influence secondary plant metabolism, but whether these effects add up when plants are co-inoculated is unclear. This issue was assessed, ...under field conditions, by comparing the early impacts of seed inoculation on secondary metabolite profiles of maize at current or reduced mineral fertilization levels. Methods Maize seeds were inoculated singly with selected strains from bacterial genera Pseudomonas and Azospirillum or mycorrhizal genus Glomus, or with these strains combined two by two or all three together. At 16 days, maize root methanolic extracts were analyzed by RP-HPLC and secondary metabolites (phenolics, flavonoids, xanthones, benzoxazionoids, etc.) identified by LC/MS. Results Inoculation did not impact on plant biomass but resulted in enhanced total root surface, total root volume and/or root number in certain inoculated treatments, at reduced fertilization. Inoculation led to qualitative and quantitative modifications of root secondary metabolites, particularly benzoxazinoids and diethylphthalate. These modifications depended on fertilization level and microorganism(s) inoculated. The three selected strains gave distinct results when used alone, but unexpectedly all microbial consortia gave somewhat similar results. Conclusions The early effects on maize secondary metabolism were not additive, as combining strains gave effects similar to those of Glomus alone. This is the first study demonstrating and analyzing inoculation effects on crop secondary metabolites in the field.
Plant-beneficial effects of bacteria are often underestimated, especially for well-studied strains associated with pathogenicity or originating from other environments. We assessed the impact of seed ...inoculation with the emblematic bacterial models Agrobacterium tumefaciens C58 (plasmid-cured) or Escherichia coli K-12 on maize seedlings in nonsterile soil. Compared with the noninoculated control, root biomass (with A. tumefaciens or E. coli) and shoot biomass (with A. tumefaciens) were enhanced at 10 days for 'PR37Y15' but not 'DK315', as found with the phytostimulator Azospirillum brasilense UAP-154 (positive control). In roots as well as in shoots, Agrobacterium tumefaciens and E. coli triggered similar (in PR37Y15) or different (in DK315) changes in the high-performance liquid chromatography profiles of secondary metabolites (especially benzoxazinoids), distinct from those of Azospirillum brasilense UAP-154. Genome sequence analysis revealed homologs of nitrite reductase genes nirK and nirBD and siderophore synthesis genes for Agrobacterium tumefaciens, as well as homologs of nitrite reductase genes nirBD and phosphatase genes phoA and appA in E. coli, whose contribution to phytostimulation will require experimental assessment. In conclusion, the two emblematic bacterial models had a systemic impact on maize secondary metabolism and resulted in unexpected phytostimulation of seedlings in the Azospirillum sp.-responsive cultivar.
Nitrogen-fixing plant growth-promoting rhizobacteria (PGPR) from the genus Pseudomonas have received little attention so far. In the present study, a nitrogen-fixing phytohormone-producing bacterial ...isolate from kallar grass (strain K1) was identified as Pseudomonas sp. by rrs (16S ribosomal RNA gene) sequence analysis. rrs identity level was high with an uncharacterized marine bacterium (99%), Pseudomonas sp. PCP2 (98%), uncultured bacteria (98%), and Pseudomonas alcaligenes (97%). Partial nifH gene amplified from strain K1 showed 93% and 91% sequence similarities to those of Azotobacter chroococcum and Pseudomonas stutzeri, respectively. The effect of Pseudomonas strain K1 on rice varieties Super Basmati and Basmati 385 was compared with those of three non-Pseudomonas nitrogen-fixing PGPR (Azospirillum brasilense strain Wb3, Azospirillum lipoferum strain N4 and Zoogloea strain Ky1) used as single-strain inoculants. Pseudomonas sp. K1 was detected in the rhizosphere of inoculated plants by enrichment culture in nitrogen-free growth medium, which was followed by observation under the microscope as well as by PCR using a rrs-specific primer. For both rice varieties, an increase in shoot biomass and/or grain yield over that of noninoculated control plants was recorded in each inoculated treatment. The effect of Pseudomonas strain K1 on grain yield was comparable to those of A. brasilense Wb3 and Zoogloea sp. Ky1 for both rice varieties. These results show that nitrogen-fixing pseudomonads deserve attention as potential PGPR inoculants for rice.
The prevalence of bacteriophages was investigated in 24 strains of four species of plant growth-promoting rhizobacteria belonging to the genus AZOSPIRILLUM: Upon induction by mitomycin C, the release ...of phage particles was observed in 11 strains from three species. Transmission electron microscopy revealed two distinct sizes of particles, depending on the identity of the Azospirillum species, typical of the Siphoviridae family. Pulsed-field gel electrophoresis and hybridization experiments carried out on phage-encapsidated DNAs revealed that all phages isolated from A. lipoferum and A. doebereinerae strains had a size of about 10 kb whereas all phages isolated from A. brasilense strains displayed genome sizes ranging from 62 to 65 kb. Strong DNA hybridizing signals were shown for most phages hosted by the same species whereas no homology was found between phages harbored by different species. Moreover, the complete sequence of the A. brasilense Cd bacteriophage (ΦAb-Cd) genome was determined as a double-stranded DNA circular molecule of 62,337 pb that encodes 95 predicted proteins. Only 14 of the predicted proteins could be assigned functions, some of which were involved in DNA processing, phage morphogenesis, and bacterial lysis. In addition, the ΦAb-Cd complete genome was mapped as a prophage on a 570-kb replicon of strain A. brasilense Cd, and a region of 27.3 kb of ΦAb-Cd was found to be duplicated on the 130-kb pRhico plasmid previously sequenced from A. brasilense Sp7, the parental strain of A. brasilense Cd.
The effect of direct inoculation of seeds with the plant growth promoting rhizobacteria (PGPR) Azospirillum lipoferum CRT1 was assessed on maize (Zea mays) grown for 35 days after sowing (d.a.s.) in ...controlled conditions (greenhouse) in a luvisol soil from south-eastern France. WhinRhizo® software was used to describe the following changes in the root system morphology for each plant: distribution and average root diameter, root surface and the number of tips. The stress at breakage and stiffness of the roots in tension were also determined. Evaluation of biochemical components of roots was achieved by direct Attenuated Total Reflectance (or reflection) (ATR)-Fourier transform infrared (FTIR) on root section. Inoculated roots exhibited significantly larger numbers of tips and extending surface to rhizosphere when compared to controls. Measured mechanical parameters of inoculated roots showed a slight increase in rupture stress up to the largest diameter (1.2 mm) when compared to controls. Stiffness (Young's modulus) values were nearly constant for inoculated plants with higher values than for non-inoculated plants at day 26 and day 35. Using Principal Components Analysis of ATR-FTIR profiles, the polysaccharide enrichment of inoculated roots compared to controls was found at day 35. Noticeable absorbance at wavenumber specific to aromatic ether (lignin) was observed in control plants. All these data had a pattern of immature root properties, when maize was inoculated with Azospirillum lipoferum CRT1. Observed modifications of root development are possibly conducive to unseen beneficial effects, like water retention, resistance to mechanical stress, or root litter quality. Studies on more mature plants are required to assess if the differences between inoculated and control plants would persist or become accentuated with time until harvest.
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