seedlings. P2b-2R endophytically colonized stem and root of cedar seedlings with a
population size of 10^4 – 106 cfu/g fresh tissue and needles with 10^1 – 102 cfu/g fresh
tissue.
Young willow (Salix sitchensis) trees growing in Three Forks Park alongside the
Snoqualmie river in Western Washington state, USA were also evaluated for the
presence of diazotrophic bacterial endophytes by Doty et al. ( 2009 ). Ten endophytic
strains capable of growing on the N-free medium were isolated and were identified
as belonging to the genera Acinetobacter, Herbaspirillum, Pseudomonas,
Sphingomonas, andStenotrophomonas. Two of the willow isolates,Herbaspirillum
sp. WW2 andPseudomonassp. H9zhy (WW6), reduced acetylene to ethylene in
acetylene reduction assay and it was also observed thatPseudomonassp. H9zhy
(WW6) possessnifgenes necessary to encode nitrogenase enzymes (Doty et al.
2009 ). These willow isolates along with some cottonwood isolates (Doty et al.
2005 , 2009 ) were tested for their ability to promote the growth of a distinct host,
Douglas-fir. Khan et al. ( 2015 ) prepared an endophytic consortium by mixing these
endophytic isolates. Endophytic consortium was inoculated into Douglas-fir and
grown in a greenhouse environment for 15 months. Inoculated seedlings had 48%
greater biomass and 13% greater root length and were 16% taller than control
seedlings. Two endophytic isolates, Acinetobacter calcoaceticus WP19 and
Rahnellasp. WP5, were tagged with GFP to visualize endophytic colonization sites
in Douglas-fir (Khan et al. 2015 ). Intercellular colonization of Douglas-fir root
tissues by WP19 and needle tissues by WP5 was observed 3 weeks after inocu-
lation. These results indicate that willow and cottonwood bacterial endophytes not
only colonize the internal tissues of a distinct host (Douglas-fir) but also promote its
growth significantly in a greenhouse environment. Such studies increase our
understanding about the bacterial endophytes that could be valuable for increasing
production of seedlings in forest nurseries.
More than two decades ago, Brooks et al. ( 1994 ) evaluated the role of endo-
phytic bacteria in suppressing oak wilt of live oaks (Quercus fusiformis). Mature
live oaks (50–70 years old) growing in sites located near Round Rock, La Grange,
and Kerrville State Park areas of central Texas, USA were sampled for bacterial
endophytes. After obtaining 889 endophytic isolates from the sapwood of live oaks,
bacteria were screened for in vitro inhibition ofCeratocystis fagacearumfungus. C.
fagacearumcauses vascular disease of oaks, commonly known as oak wilt. The
traditional method of eradicating oak wilt is to remove diseased trees from the site
and break the connections between the healthy and diseased tree (Gibbs and French
1980 ; MacDonald and Hindal 1981 ) or injecting a fungicide into the intravascular
tissues of the oak plant (Appel and Kurdyla 1992 ). Brooks et al. ( 1994 ) hypothe-
sized that biological control through endophyte inoculation could be a possible and
sustainable way of controlling oak wilt. Six endophytic isolates belonging to genera
BacillusandPseudomonaswere screened after in vitro evaluation of their ability to
suppress C.fagacearum. When injected into the stem,Pseudomonasspp. exten-
sively colonized live oaks. The ability ofPseudomonasstrains to control the oak
wilt pathogen in vivo was evaluated in two growth trials. Inoculation decreased the
number of trees diseased by 50% and reduced the crown loss by 17%. This study
126 A. Puri et al.