after inoculation, respectively (Table6.2). In a subsequent study, Anand et al.
( 2013 ) reported that lodgepole pine seedlings inoculated with P2b-2R were able to
derive 79% of N directly from the atmosphere, 13 months after inoculation
(Table6.2). Along with fixing high amounts of N, P2b-2R inoculation also
enhanced lodgepole pine shoot height by 33%, shoot dry weight by 78%, and root
dry weight by 165%. They postulated that plant growth promotion was directly
related to the amount of Nfixed by P2b-2R. Since seedlings were grown in an
N-limited environment and N fertilizer was provided only once at the onset of the
experiment, so after sometime, soil N depletion would eventually restrict the growth
rate of control seedlings to a point where P2b-2R inoculated (N-fixing) seedlings
would outperform them. When lodgepole pine seedlings were grown in sufficient N
conditions (N fertilizer provided regularly in a yearlong growth experiment),
P2b-2R inoculation had no effect on the growth of lodgepole pine and the inocu-
lated seedlings were not able tofix atmospheric N (Yang et al. 2016 , 2017 ), thus
confirming the hypothesis proposed by Bal and Chanway (2012a) that P2b-2R
triggers Nfixation mechanism under N-limited conditions. Full sequencing of
nitrogenase reductase protein (nifH) of P2b-2R was also conducted by amplifying a
388-bp internalnifH gene fragment and performing a Southern blot analysis of total
genomic DNA digested with Pst I/HindIII (Anand and Chanway2013c). The
Southern blot profile showed just one positive signal at 1.8 kb of the Pst I digest
and 9 kb of the HindIII digest (Fig.6.1), indicating that there is only one copy of
nifH in P2b-2R. Anand et al. ( 2013 ) evidenced that P2b-2R strain can form a
significant amount of endophytic colonies in the root, shoot, and needle tissues,
thus indicating that perceived growth promotion and Nfixation was bacteria driven.
But in this study, a culture-based technique was used to assess the bacterial colonies
in each part of the plant. Endospore-forming bacteria likeP. polymyxaP2b-2R are
susceptible to misidentification when such technique is used (Bent and Chanway
2002 ). Anand and Chanway (2013a) then applied a more precise technique to prove
that P2b-2R can colonize lodgepole pine endophytically. Greenfluorescent protein
(GFP) tagging in conjunction with confocal laser scanning microscopy (CLSM)
was used to view the sites of endophytic colonization in real time. A plasmid-borne
GFP (pBSGV104) was used to tag P2b-2R and the transformed strain was named
Table 6.2 Percent N derived from the atmosphere (%Ndfa) by a diazotrophic bacterial
endophyte,Paenibacillus polymyxaP2b-2R, when inoculated into two different host trees and
determined in several studies at different time periods
Host tree Time after inoculation %Ndfa References
Lodgepole pine 27 weeks 30 Bal and Chanway (2012a)
35 weeks 66 Bal and Chanway (2012a)
12 months 40 Tang et al. ( 2017 )
13 months 79 Anand et al. ( 2013 )
Western red cedar 27 weeks 23 Bal and Chanway (2012b)
35 weeks 56 Bal and Chanway (2012b)
13 months 36 Anand and Chanway (2013b)6 Beneficial Effects of Bacterial Endophytes... 117