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

74j 4 A Review on the Taxonomy and Possible Screening Traits of Plant Growth Promoting Rhizobacteria
[102]. In 1958, nitrogen fixation was conclusively proven for a Bacillus strain, later
identified as B. polymyxa (now Paenibacillus polymyxa) [102]. Classically, all Bacillus
strains capable of fixing molecular nitrogen were found to belong to B. polymyxa or B.
macerans(nowPaenibacillusmacerans),aswellassomestrainsidentifiedasB.circulans,
until Seldin et al. [103] described B. azotofixans (now Paenibacillus durus [104]).
Achouak et al. [105] made a comparative phylogenetic study of 16S rDNA and nifH
genes in the family Bacillaceae, and concluded that nitrogen fixation among aerobic
endospore-forming bacteria is restricted to the genus Paenibacillus. To date, nitrogen
fixation has been demonstrated for 11 species of Paenibacillus: P. polymyxa, P. macerans,
P. durus, P. peoriae, P. borealis, P. brasilensis, P. graminis, P. odorifer [106], P. wynnii
[106], P. massiliensis [107] and P. sabinae [108]. Strains belonging to these species act as
plantgrowthpromotingrhizobacteriabecauseoftheirN2-fixationability,productionof
phytohormones, provision of nutrients and/or by the suppression of deleterious microorganisms
through antagonistic function [109].
4.5
Screening Methods of PGPR
4.5.1
Culture-Dependent Screening Methods
Traditionally, a search for PGPR involves screening a large number of isolates and
identifying a desired phenotypic trait. Once isolates are purified, the main goal is to
maintain the maximum genetic diversity in the minimum number of isolates for
identifying the desired phenotypic trait or for performing further biological assays in
order to achieve results that are representative of the diversity occurring in Nature.
This goal may be achieved through the use of intergenic transcribed sequence (ITS)-
PCR, AFLP and arbitrarily primed (AP)-PCR/PCR–RAPDs techniques that define
differences at the strain level [10,110].
Once the set of strains to test is defined, the screening methods used will differ
accordinglytothetraitofrelevanceinthestudy.Screeningmethodshavebeendescribed
forthedetectionofarangeofsinglemolecules(e.g.auxinsproductionbycolorimetry[6])
or an array of characteristic traits of the PGPR (e.g. screening for PGPR by testing for
aminocyclopropanecarboxylic acid (ACC), auxin and siderophoreproduction and phosphate
solubilization [10]). Three rapid plate assays have been developed that allow for
screening of those PGPR capable of inducing plant-systemic resistance based on their
ability to attack certain plant-pathogenic fungi [89] by targeting the fungal pathogenesisrelated
proteins chitinase and b-1,3-glucanase,and their biphasichydrogen production.
Other screening techniques for isolated strains rely on molecular methods to
search for diverse marker genes, such as the (ACC) deaminase gene [23], or genes
associated with plant–LNB interactions, such as the nodA [110] or gusA and celB
genes. The marker genes gusA and celB were used to study plant–Rhizobium interactions,
and the competition within inoculated strains and between inoculated
strains and indigenous rhizobia. A GUS Gene Marking Kit has also been developed