- Bacillus subtilis strain B26 is an endophytic bacterium that promotes plant growth in timothy grass. - Experiments showed bacterized timothy grass had longer, more complex roots and accelerated flowering compared to non-bacterized plants. B26 successfully colonized inside timothy grass tissues. - Exposure to B26 volatile organic compounds significantly increased root, shoot, and total biomass of timothy grass seedlings compared to controls. A B26 culture filtrate amendment to growth media also enhanced total biomass of timothy grass at concentrations of 500 and 1000ppm.

1. Master Sciences de l’Univers, Environnement, Ecologie
M1 2013-2014
Fig 6: At 17dpi, significantly higher dry mass of
plants grown in a culture media amended with
500 and 1000ppm was observed. (p<0.05)!
Fig. 1: At 28dpi, visual evidence supports
that bacterized plants (left) developed a
longer and more complex rooting system.!
Fig. 3: At 21dpi, plants
exposed to B26 (top right)
and B168 (top left) VOCs
were visually more
developed in both their
rooting system and
aboveground biomass.
(p<0.05)!
!
Table 1: Log CFU at 21dpi!
INTRODUCTION!
A strain of Bacillus subtilis B26, recently reported as an
endophytic bacterium of the bioenergy crop switchgrass, is
nonpathogenic and a growth enhancer. !
!
Culture-filtrate from B26 contains several well-
characterized lipopeptide toxins and phytohormones. These
qualities suggest that endophytic ability of this strain is a
biological requirement for survival in nature and has
strong potential as bio-inoculant for biomass enhancement
of bioenergy and forage crops, and enhancement of plant’s
defense against abiotic stress such as drought stress. !
!
Timothy grass (Phleum pretense) is a major forage crop
widely used in Quebec, in combination with Alfalfa.
Drought as well as high temperatures impair the
performance of Timothy, and thus makes it a good
candidate for investigating the enhancement of hydric
stress tolerance using B. subtilis strain B26.!
!
!
!
CONCLUSIONS!!
• A single inoculation of Timothy grass
seedlings with B. subtilis B26 exerted
phenotypic effects in the root system (Fig.
1). Bacterized plants had accelerated
flowering time. These results confirm B26
growth promoting abilities.!
• Successful internal colonization of tissues
of Timothy grass grown under gnobiotic
and growth chamber conditions by B.
subtilis B26 confirms that B26 is a
competent endophyte (Table 1, Fig.2).!
• Growth promotion by VOCs was more
effective with B26, suggesting it is not
specific to all endophytes. It is not yet
known if this effect is mediated through a
single or a blend of volatiles, which already
have been characterized (Fig.3-5).!
!
• Bioactive filtrate of B26 at 1000 ppm
enhanced total biomass of Timothy grass
seedlings (Fig. 6).!
• Future studies will aim at developing a
biofertilizing method using B26 biofiltrate
to reduce water and fertilizer use, avoiding
soil ecology modifications associated with
bacterial amendments.!
!
Camille Ziegler1, François Gagné-Bourque2, Suha Jabaji2!
1Master Ecophysiologie et Ecotoxicologie (EPET), Université Pierre & Marie, Paris, France!
2Department of Plant Science and Agriculture, Macdonald Campus of McGill University, Ste-Anne-de-Bellevue, QC, Canada!
!
METHODS!
AKNOWLEDGEMENTS!RESULTS!
OBJECTIVES!!
• Confirm the internal and systemic colonization of
Timothy grass tissues by B. subtilis B26!
• Examine the ability of B. subtilis B26 to promote
vegetative growth of Timothy grass!
• Determine whether bioactive extract of B. subtilis B26
enhances growth !
*! *!
*! *!
*! *!
0"
2"
4"
6"
8"
10"
12"
14"
B168! B26! Control! DH5"!
Lenght(cm)!
Treatment!
Root, shoot and total length of Timothy
exposed to different bacterial VOC!
Root!
Shoot!
Total!
*"
0"
1"
2"
3"
4"
5"
6"
7"
8"
9"
10"
B168! B26! Control! DH5"!
Dryweight(mg)!
Treatment!
Timothy dry mass exposed to different
bacterial VOC!
Enhancing Timothy grass (Phleum pratense) growth through
endophytic bio-inoculant Bacillus subtilis B26
REFERENCES!!
!
F. Gagné-Bourque et al. (2012): Isolation
and characterization of indegenous
endophytic bacteria associated with leaves
of switchgrass (Panicum virgatum L.)
cultivars. Journal of Applied Microbiology
ISSN 1364-5072!
!
Fig 5: At 21dpi,
plants exposed to
VOCs from B26 had
a significantly
higher dry weight
than control plants.
(p<0.05)!
Fig 4: At 21dpi,
seedlings exposed
to B26 and B168
V O C s w e r e
significantly larger
than controls.
(p<0.05)!
!
Fig 2: Internalization of B26 is confirmed by PCR in Timothy tissues.!
Source! Average /gram ± SD!
Root! 2.77±2.15!
Shoot! 2.52±1.92!
Soil! 5.56±5.21!
Soil-grown ! In-vitro!
B+/S B+/R! B-/S B-/R! B+/S B+/R! B-/S B-/R!
565bp!
+VE! -VE!
*"
*"
0"
0.2"
0.4"
0.6"
0.8"
1"
1.2"
1.4"
1.6"
1.8"
2"
1000" 500" 250" 0"
Dry%weight%(mg)%
Extract%concentra5on%(PPM)%
Timothy%dry%weight%%
LB" LB"+"extract"
Range of concentration !
0  10000ppm!
B-! B+!
B. subtilis (B26) culture! B26 bioactive extract!
B26!
Control!
E. Coli
DH5"!
B168!
B+!B-!
• Inoculation D10!
• Harvest 23dpi*!
• Inoculation D14!
• Harvest 6dpi, 8dpi, 15dpi,
28dpi!
• Harvest 21dpi!
• Harvest 17dpi!
Ⅰ. In vitro! Ⅱ. Soil grown! Ⅳ. Extract!Ⅲ. Volatiles!
Culture independent
(PCR)! Culture dependent !
(CFU & internal colonization)!
Agronomical measurements!
germination, root and shoot length, dry mass!
* days post inoculation!
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