Page 2 Plant-Bacteria Interactions Edited by Iqbal Ahmad, John ...

90j 5 Diversity and Potential of Nonsymbiotic Diazotrophic Bacteria in Promoting Plant Growth
nitrogen may be derived from free-living nitrogen-fixing cyanobacteria in floodwater
or heterotrophic nitrogen fixers in the soil [94]. To provide direct evidence that the
plant benefits from the nitrogen fixed by the assumed diazotroph, plant inoculation
experiments with nonnitrogen-fixing (Nif ) mutants as negative controls are
required, coupled with careful 15 N-based balance studies. With the use of such
mutants in inoculation experiments, it becomes clear that, in most cases, BNF is
not involved in plant growth promotion. Nif mutants of Azospirillum, Azoarcus sp.
strain BH72 or Pseudomonas putida GR12-2 have been shown to still be capable of
stimulating plant growth [95,96]. No 15 N isotope dilution or nitrogen balance
experiments have been carried out with these Nif mutants. The fact that BNF is
apparently not involved in plant growth promotion by these strains cannot be simply
attributed to the absence of nitrogenase expression. Using a translational nifH–gusA
fusion, it was observed that Azospirillum nif genes are expressed during the association
with wheat roots [97]. However, some host specificity of BNF has been reported;
for example, when the nifK mutant of Azoarcus sp. strain BH72, which has a Nif
phenotype, was used to inoculate rice seedlings in a gnotobiotic system, the same
increase in plant biomass and total protein content was found as after inoculation
with the wild-type strain, strongly suggesting that nitrogen fixation was not involved
in the observed plant growth promotion [96]. Nevertheless, immunogold labeling as
well as reporter gene studies revealed high nitrogenase gene expression levels of the
endophyte Azoarcus sp. BH72 inside roots of rice seedlings, suggesting that environmental
conditions inside rice roots will allow endophytic nitrogen fixation in bacterial
microcolonies in the aerenchyma [98]. However, when this Nif mutant was
inoculated onto Kallar grass plantlets, these plants showed significantly lower dry
weight and accumulated less nitrogen than those inoculated with the wild-type strain
Azoarcus sp. BH72 [99,100]. The associative symbiosis (using Azospirillum) was
observed in paranodules of maize and nonnodulated maize plants. An increase in
nitrogenase activity (acetylene reduction assay) and leghemoglobin content was
observed in plants treated with Azospirillum [101]. The five bacterial isolates (two
of Stenotrophomonas maltophilia, two of Bacillus fusiformis and one of Pseudomonas
fluorescens) were showing nitrogenase activity above 150 nmol h 1 mg 1 protein
[102]. Three bacterial species of Bacillus could fix nitrogen and significantly
increased the growth of barley seedlings [103].
5.4
Plant Growth Promoting Mechanisms of Diazotrophic PGPR
The plant growth promoting rhizobacteria may promote plant growth either directly
or indirectly. Direct mechanisms include (i) the ability to produce plant growth
regulators (indole acetic acid, gibberellins (GAs), cytokinins (CTKs) and ethylene)
[91,104,105] and (ii) solubilization of mineral nutrients such as phosphates
[106–109]. Indirect mechanisms involve (i) antagonism against phytopathogens
[110,111], (ii) production of siderophores [110,112], (iii) production of extracellular
cell wall degrading enzymes for phytopathogens, for example b-1,3-glucanase [113]