P2b-2Rgfp. With the help of CLSM, it was observed that P2b-2Rgfphad com-
pletely engulfed the root surface of lodgepole pine, similar to what was reported by
Timmusk et al. ( 2005 ). P2b-2Rgfpeffectively colonized stem cortical cell of 2–
14-week-old lodgepole pine seedlings intracellularly (Figs.6.2 and 6.3) but was not
observed in vascular tissues. Thus, Anand and Chanway (2013a) provided a strong
evidence that P2b-2R can effectively colonize stem tissues endophytically from as
early as 2 weeks after inoculation. However, Padda et al. (2016a) reported that
GFP-tagging might affect the performance of P2b-2R to promote plant growth and
fix N. After some successful initial reports about colonization and plant growth
promotion of agricultural crops, viz., corn, canola, and tomato by P2b-2R. Puri
et al. ( 2015 , 2016a), Padda et al. (2016a, 2017) reported that GFP-tagging could
positively affect the plant growth-promoting and N-fixing capability of P2b-2R in
agricultural crops like corn and canola. Recently, Puri et al. (2016b) concluded that
this effect is temporary in corn and diminishes as the plant develops. However,
Padda et al. (2016b) further reported that the positive effect of GFP-tagging is not
temporary in canola and could be seen throughout the life cycle of the plant. Tang
et al. ( 2017 ), in an effort to evaluate the positive effect of GFP-tagging of P2b-2R in
its original host lodgepole pine, found that P2b-2Rgfpoutperforms the wild-type
strain in the initial stages of plant development only (till 4 months after sowing) and
the positive effect diminishes as the pine plant grows. It has been discovered that
GFP-tagging leads to overexpression ofnifH,nifD, andnifK genes. Therefore, the
effect of reporter gene GFP on physiological activities of host–microbe cell should
be taken into account in using it as a cytological marker (Unpublished data).
Fig. 6.1 Southern blot profile ofP. polymyxaP2b-2R total DNA digested with Pst I (Lanes 1, 2 ,
and 4 ) or HindIII (Lanes 5, 6 , and 8 ) and probed with thenifH fragment (from Anand and
Chanway2013c)
118 A. Puri et al.