tmpEAC2 TMP
tmpEAC2 TMP
DOI 10.1007/s11104-009-9908-1
REGULAR ARTICLE
DO09908; No of Pages 10
Plant Soil
selected (Table 1), grown in MG/L medium overnight, and stored in 33% glycerol at 80C for
subsequent characterization. To identify the role of
sweet potato endophytes in plant growth, we generated some internally sterile plants by propagating
them in MS medium containing antibiotics. The
concentration of the antibiotics used was determined
from previous experiments (data not shown) that was
enough to kill the endophytes and not harm the plant.
Validation of sterility was done by crushing the plants
in sterile conditions and growing the extract on MS
plates and checking for bacterial growth.
Bacteria identification using 16S rRNA sequences
Genomic DNA was prepared using standard methods
(Ausubel et al. 1995) with a cell lysis performed at
68C for 30 min (Doty et al. 2005). PCR was
performed using the universal 16S rRNA primers 8F (5- AGAGTTTGATCCTGGCTCAG-3) and
1492 (5- GGTTACCTTGTTACGACTT-3) as described previously (Doty et al. 2005). The thermocycler program was 94C for 15 s, 44C for 15 s,
72C for 30 s and it was repeated for 24 cycles.
Samples were subjected to electrophoresis in a 1%
agarose gel. The 1.5 Kb PCR products were purified
using Qiagen gel extraction kit (Qiagen; Valencia,
CA) subcloned into pGEM T Easy (Promega;
Madison, WI) and sequenced using T7 and SP6
primer sets on the vector by the University of
Washington Biochemistry Department Sequencing
Facility using the Big Dye Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems; Carlsbad, CA)
Table 1 Characteristics of isolated endophytes from sweet
potato
Endophyte code
Plant
Colony color
on MG/L plates
SPa
SPb
SPc
SPd
SPe
SPf
SPg
SPh
SPi
SPj
SPk
SP3
SP3
SP2
SP4
SP3
SP4
SP3
SP3
SP1
SP2
SP4
White
Pink
White
White
Pale yellow
White
White
White
Yellow
White
White
Plant Soil
98
99
100
SPi (FJ405368)
AM419020 Rahnella sp. NJ-8
95
99
SPb (FJ405361)
97
100
SPe (FJ405364)
100
100
99
Spa (FJ405360)
SPd (FJ405363)
100
SPc (FJ405362)
0.02
Plant Soil
Plant Soil
Results
Identification
0.3
0.25
0.2
0.15
0.1
0.05
0
0
0.5
1.5
2.5
Time in days
AZ
AG
Spa
SPb
Plant Soil
371bp
that drastically modify all physico-chemical parameters of a living cell. To test whether IAA production
affected cell viability at temperatures that normally
prevent growth, diluted cultures were plated on agar
plates and inoculated at different temperatures.
Isolate SPb survived cold treatment whereas SPh
did not grow. The UV survival analysis showed that
the IAA producing endophyte was more resistant
(more CFU) to UV irradiation compared to the
negative control. Finally, responses to heat shock,
low pH, osmolarity and oxidative damage were
similar (Table 2).
Discussion
Endophytes isolated from four sweet potato plants
exhibited a wide range of phenotypic diversity and
important functions. Growth in nitrogen free media,
and the presence of nifH sequences verified in one of
the isolate supports the hypothesis that the sweet
potato endophytes may be of diazotrophic nature.
Further studies are needed to determine if the
endophytes might contribute to the N input in sweet
potato plants that grow well in unfertile soil.
Previous reports indicated that associative N2 fixation contributes to the N uptake in sweet potato
plants (Yoneyama et al. 1998; Hill et al. 1990).
Nitrogen fixing endophytes like Azospirillum sp.
Indole acetic acid (IAA) production by sweet potato endophytes
0.5
0.45
Stress tests
Absorbance at 530nm
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
SPb
Spe
Spi
SPj
Plant Soil
Fig. 5 Root formation by
an IAA producing endophyte SPb in a hybrid poplar clone. When inoculated
into INRA 7171B4, four
out of the five plants
showed root formation and
increased plant growth
when compared to uninoculated controls
(Hill et al. 1983) and Gluconacetobacter diazotrophicus strains (Paula et al. 1991) were isolated from
root and aerial part of sweet potato plants, respectively. Reiter et al. (2003) identified potential
nitrogen fixing endophytes in seven sweet potato
varieties collected in Uganda and Kenya. The nifH
gene sequences had high homologies to the nitrogenase reductases of known nitrogen fixing bacteria.
The authors suggested that the presence of these
bacteria might contribute to the N input in sweet
potato plants grown in different soils on small scale
farms in Africa where no fertilizer is applied. In our
study since Spa showed the presence of nitrogenase
gene, we are currently doing experiments to investigate if it could help the sweet potato plant to grow
in nitrogen limited conditions.
Some of the isolated endophytes of sweet potato
synthesized the phytohormone IAA, which is involved in plant stem and root growth regulation. It is
the most potent native auxin and is synthesized by
plants from the amino acid tryptophan. The capacity
Table 2 Resistance of SPb (IAA producer) and SPh (IAA non-producer) isolates to various stress conditions and antibiotics
Endophytes selected
SPb
SPh
Cf
Tm
Km
Ap
Cm
Cb
Vm
4C
UV exposure (CFU)
3102
1.5102
3.5102
3.6102
Heat shock
+
+
pH
NaCl
H2O2
(3M)
(3%)
+
+
+
+
+
+
+
+
Plant Soil
Plant Soil
Appendix
Partial SS
df MS
Model
11.7247352
19 .617091327 75.98
Sample
8.51532782
3 2.83844261 122.51
Flask|Sample
.185357703 8 .023169713
Time
2.3544058
2 1.1772029
144.94
Time*Sample
.669643897 6 .111607316 13.74
Residual
.129953838 16 .008122115
Total
11.8546891
35 .338705402
Prob > F
0.0000
0.0000
0.0000
0.0000
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