geographical area as the bacteria (Shishido and Chanway 1999 ). In an interesting
study, Shishido and Chanway ( 2000 ) used a combination of greenhouse andfield
trial to assess the growth-promoting effects of Ss2-RN. Hybrid spruce seedlings
werefirst grown in greenhouse for 4 months and were then outplanted infield sites.
Some seedlings were harvested after greenhouse trial to assess the growth pro-
motion due to inoculation of Ss2-RN in thefirst 4 months. As expected, Ss2-RN
inoculated seedlings had significantly higher root and shoot biomass than controls.
Relative growth rate (RGR) of outplanted seedlings was determined 4 months after
outplanting in thefield. Root and shoot RGR of inoculated seedlings were 10–
234% higher than controls, thus establishing the fact that bacterial endophyte
Ss2-RN can perform exceptionally infield conditions. Surprisingly, Ss2-RN was
not able to colonize spruce seedlings endophytically as observed in two different
studies (Shishido and Chanway 2000 ; Chanway et al. 2000 ), thus indicating that
this strain promotes spruce tree growth by colonizing the rhizosphere.
Shishido and Chanway ( 1999 ) had also isolated a PGPR,PseudomonasSm3,
from rhizosphere of 1–3-year-old hybrid spruce seedlings naturally regenerating
near Mackenzie, BC, Canada (55°11′N, 122°58′W, 780 m elevation). Sm3-RN, a
rifamycin-resistant derivative of Sm3, was found to colonize rhizosphere of spruce
seedlings with a population density of 10
4
- 10
5
cfu/g rhizosphere soil and signifi-
cantly enhance root and shoot weight and seedling height (Shishido et al.1996b).
Although Sm3-RN was isolated from rhizosphere, it was able to colonize root
interior of hybrid spruce seedlings grown in both greenhouse andfield conditions
with a population size of 10^2 – 104 cfu/g root tissue and significantly promote root
and shoot RGR when grown at afield site (Shishido and Chanway 2000 ). Internal
root tissue colonization and growth promotion of spruce seedlings by Sm3-RN in
field conditions was also confirmed by Chanway et al. ( 2000 ). Apart from
culture-based studies, Shishido et al. ( 1999 ) also evidenced the endophytic colo-
nization by Sm3-RN of spruce seedlings by using immunofluorescent antibody
staining (IFAS) technique. Sm3-RN strain was detected in root hairs, cortical cells,
and stem vascular tissues of spruce after 4 months of inoculation. It can be con-
cluded that Sm3-RN, a PGPR, enters the spruce seedling likely through the root
openings and form detectable endophytic colonies in root and stem tissues.
A lodgepole pine endophyte,Paenibacillus polymyxaPw-2R (Shishido et al.
1995 ), was also tested for its ability to endophytically colonize hybrid spruce and
promote its growth. Strain Pw-2R was able to colonize internal root and stem
tissues of hybrid spruce (Picea glaucaxengelmannii) with population size of 10
4
10
5
cfu/g root tissue in controlled environment experiment 5 months after inocu-
lation and promoted seedling biomass by 57% infield trials 17 months after
inoculation (Chanway et al. 2000 ). Effects of Pw-2R inoculation on spruce seed-
lings were also assessed by Shishido et al. (1996a). Endophytic colonization of
hybrid spruce by culture-based technique was also evaluated by Shishido and
Chanway ( 2000 ) and it was observed that this endophyte can colonize internal root
tissues with a population size of 10
2
- 10
4
4 months after inoculation and increase
shoot RGR by up to 82%. Internal tissues colonization of spruce by Pw-2R was also
evaluated by IFAS technique to have a more precise evidence since culture-based
124 A. Puri et al.