FEMS Microbiology Ecology 13 (1994) 249-258
© 1994 Federation of European Microbiological Societies 0168-6496/94/$07.00
Published by Elsevier
249
FEMSEC 00505
Effect of a genetically modified
Pseudomonas aureofaciens on indigenous microbial
populations of wheat
F r a n s A . A . M . d e L e i j *, E m m a
J. S u t t o n , J o h n M . W h i p p s a n d J a m e s M . L y n c h 1
Microbiology and Crop Protection Department, Horticulture Research lntemationa~ Worthing Road, Littlehampton, West Sussex,
BN17 6LP, UK
(Received 23 August 1993; revised 2 November 1993; accepted 16 November 1993)
Abstract: An isolate of Pseudomonas aureofaciens from the phylloplane of sugar beet which was chromosomally modified for
monitoring purposes by the insertion of two gene cassettes (kmr-xylE and lacZY) was introduced to the phytosphere of spring
wheat in a number of experiments and the resulting microbial perturbations quantified. Such studies involving innocuous bacterial
isolates can serve as a guide in the assessment of risk associated with the release of functionally modified microorganisms.
Introductions of P. aureofaciens on seeds caused large microbial perturbations (up to 2 log units) at the seedling stage on seeds and
roots. As the inoculated plants matured (tillering, flowering and ripening), perturbations of total microbial populations were found
to be non-significant. Microbial perturbation on maturing wheat roots as a result of seed inoculations with P. aureofaciens could
only be detected using more sensitive monitoring procedures describing the Pseudomonas community in terms of colony
appearance rate on a selective Pseudornonas medium. Spray applications of the marked P. aureofaciens isolate onto the leaf
surface of wheat caused no significant perturbations of the indigenous microbial populations present on the phylloplane.
Key words: Microbial populations; Pseudomonas aureofaciens; GEMMO; Wheat; Glasshouse; Risk
Introduction
Genetically modified microorganisms (GEMM O s ) have p o t e n t i a l for c r o p p r o t e c t i o n , imp r o v e m e n t o f soil s t r u c t u r e a n d n u t r i e n t status,
b i o r e m e d i a t i o n a n d m i n e r a l leaching. H o w e v e r ,
* Corresponding author. Tel: (0903) 716123; Fax: (0903)
726780; E-Mail: [email protected]
1 Present address: School of Biological Sciences, University
of Surrey Guildford, Surrey, GU2 5XH, UK.
$SDI 0168-6496(93)E0065-P
b e f o r e r e l e a s e o f such f u n c t i o n a l l y active r e c o m b i n a n t s into t h e e n v i r o n m e n t c a n t a k e place, a
b e t t e r u n d e r s t a n d i n g o f G E M M O s in t h e envir o n m e n t in g e n e r a l is n e e d e d [1]. T o achieve this
goal, t h r e e m a i n a r e a s c o n n e c t e d with G E M M O
i n t r o d u c t i o n have to b e a d d r e s s e d . Firstly, m o r e
i n f o r m a t i o n is n e e d e d o n t h e survival a n d d i s p e r sal o f r e c o m b i n a n t o r g a n i s m s in t h e e n v i r o n m e n t .
Secondly, i n f o r m a t i o n is r e q u i r e d o n p o s s i b l e int e r a c t i o n s o f t h e s e o r g a n i s m s with o t h e r s p e c i e s
a n d / o r b i o l o g i c a l systems in t h e e n v i r o n m e n t
w h e r e i n t r o d u c t i o n will t a k e place, i n c l u d i n g el-
250
fects of the products produced by the heterogenous genes. Thirdly, information is needed on the
transfer of introduced genetic material to indigenous organisms. The risks associated with any (or
a combination) of these factors can be defined as
the product of the likelihood of a harmful effect
and the degree of severity of such an effect [2].
However, the problem with such a risk assessment for the purpose of GEMMO introduction is
that there are no quantitative data available which
provide a measure for the severity of effects
caused by such introductions. The first step in
this should therefore consist of studies monitoring effects that an innocuous GEMMO might
have on ecosystem function.
This paper describes such a base line study, in
which a chromosomally double-marked, nonpathogenic strain of Pseudomonas aureofaciens
[3] was introduced onto wheat in the glasshouse
for the purpose of monitoring microbial perturbations.
Materials and Methods
Organisms used
The wild-type organism (SBW25) was isolated
from the phylloplane of sugar beet and was identified by fatty acid methyl ester analyses (FAMEMIDI) as a strain of P. aureofaciens (LOPAT
group IV). P. aureofaciens was one of the most
commonly occurring bacteria in soil and on roots
and leaves of both sugar beet and spring wheat,
occurring at percentages between 2 and 40% of
the total culturable bacterial populations during
the growing season [4] (F.A.A.M. de Leij and
D.E. Legard, unpublished).
By site-directed homologous recombination,
two different sets of marker genes were introduced and stably maintained into two well-separated, presumed non-essential sites of the chromosome of strain SBW25 to create the recombinant SBW25EeZY-6KX (M.J. Bailey, unpublished). The marker genes were chosen to facilitate detection by simple selective agar plating
methods and included:
(a) The lacZY genes for lactose utilisation. The
lacZY system [5-7] is one of the most widely used
metabolic markers [8] and bacteria of the genus
Pseudomonas do not utilise lactose as a carbon
source [6]. In this system, the insertion of the
Escherichia coli genes lacZ (/3-galactosidase) and
lacY (lactose permease) enables the modified organism to utilise lactose as a carbon source. When
organisms with the lacZY genes are plated onto
media containing the chromogenic substrate 5chloro-4-bromo-3-indolyl-/3-D-galactopyranoside
(X-gaD, the lacZY genes facilitate cleavage of this
substrate resulting in easily identifiable, blue
colonies.
(b) The marker cassette kmr-xylE. The km ~ gene
encodes for resistance to the antibiotic kanamycin
and was derived from Tn903. The xylE gene
originated from the TOL(pWWO) catabolic plasmid [9], and enables the conversion of its substrate catechol into a yellow compound (2-hydroxymuconic semi-aldehyde).
The expression of these two gene cassettes in
P. aureofaciens did not result in a functional
advantage for the recombinant compared with
the parent strain from which it was derived.
Cells of both wild-type and recombinant P.
aureofaciens were stored at -70° C in 20% glycerol (v/v). When required for experimental use,
cells were removed from the freezer and plated
onto Tryptic Soy Agar (TSA; 30 g Tryptic Soy
Broth (TSB; Oxoid) and 15 g technical agar
(Oxoid) in 1 1 water). After incubation for 24 h at
25°C, cells were taken from the TSA plate and
inoculated into 500-ml conical flasks, containing
100 ml sterile TSB. To obtain a bacterial culture
in late log phase, flasks were incubated overnight
on a rotary shaker (200 rpm; 28°C). Bacterial cells
were harvested by centrifugation (5000 x g, 5
rain). To remove most of the culture medium, the
bacterial pellet was resuspended and centifuged
(5000 x g, 5 min) twice in sterile 0.25 strength
Ringer's solution. For seed inoculations, a bacterial suspension containing 109-10 l° cells m1-1
was prepared.
Effect of P. aureofaciens seed inoculations on wheat
seedlings
Spring wheat seeds (cv. Axona) were vacuum
infiltrated ( - 0.2 bar) for 20 min in a suspension
of 10 ~° recombinant P. aureofaciens cells m1-1
0.25 strength Ringer's solution. After vacuum in-
251
filtration, each seed contained 8.2 +_ 1.1 x 106 recombinant cells. Three treatments were used: (a)
soaked, untreated seeds suspended in 1.25% guar
gel (Liquid Drilling Ltd., Stratford upon Avon,
UK); (b) seeds inoculated with recombinants, but
without guar gel; and (c) seeds inoculated with
recombinants suspended in 1.25% guar gel. Seeds
were planted at a depth of 2 cm in trays (35 x 25
x 5 cm deep) filled with a mixture of 75% silt
loam soil (Hamble series) and 25% grit (soil/grit)
(25 seeds per tray; four replicates per treatment).
The trays were placed on a bench at 20°C in four
randomised blocks according to replicate. Five
days after planting, emergence of wheat seedlings
was estimated, as well as the root and shoot
(fresh) weights of five randomly selected seedlings
taken from each tray.
The colonisation by Pseudomonas of seeds,
roots and leaves was estimated on the plants that
were removed from each tray. Roots were carefully washed in tap water to remove loose soil.
Each sample was divided into roots, seeds and
leaves. The roots and leaves from each sample
were cut into 1 cm pieces. Each subsample was
put in a universal bottle containing 10 ml, 0.25
strength Ringers and 0.05% (w/v) agar (RA). To
overcome the hydrophobic properties of the wheat
leaf surface, 0.1 ml of a 1% (v/v) Triton X-100
suspension was added to each universal bottle
containing leaves. After treatment for 7 rain in a
sonication bath (Kerry Ultrasonic Ltd.), roots and
leaves were removed. Seeds were crushed with a
sterile pestle and mortar in 10 ml RA to release
the microbial populations associated with the
seeds. From each sample, ten-fold dilutions were
prepared in RA. Aliquots of 0.1 ml of each dilution were spread onto a Pseudomonas selective
agar (P-l) [10] and incubated for 5 days at 25°C.
Fluorescent Pseudornonas colonies were enumerated by exposing plates to ultraviolet light (366
nm). To distinguish indigenous fluorescent Pseudomonas from recombinant P. aureofaciens
colonies, plates were flooded with a freshly made
solution of 1% (w/v) catechol. Colonies that
turned yellow (xy/E activity) were enumerated as
recombinant P. aureofaciens and could be subtracted from the total Pseudomonas count to
obtain the number of indigenous fluorescent
Pseudomonas. Total fluorescent Pseudomonas, indigenous fluorescent Pseudomonas and recombinant populations were expressed as colony forming units (cfu) per plant part.
Effect of P. aureofaciens seed inoculations on microbial populations on roots at tillering, flowering
and ripening
To estimate longer term effects of seed inoculations with P. aureofaciens, wheat seeds (ev. Axona) were inoculated with either wild-type or
recombinant P. aureofaciens by incubating overnight in a suspension of 10 l° bacterial cells ml-1
1% (w/v) methyl cellulose (MC). After inoculation 5 X 10 s bacterial cells were recovered from
each seed. The control treatment was incubated
overnight in sterile MC. Seeds were pregerminated on wet filter paper at 25°C before
planting at a depth of 1.5 cm in microcosms (five
pre-germinated seeds per microcosm; 12 microcosms per treatment; completely randomised design). Each microcosm consisted of an undisturbed soil core of silt loam soil (Hamble series;
1.4% carbon) with a weight of 18 kg, held in a
PVC tube (60 cm length and 15 cm diam.). The
microcosms had been stored vertically in a shallow trench in the field for a year before they were
transferred to an unheated, well-ventilated,
glasshouse where they were placed in wooden
boxes to provide insulation from direct sunlight
[11]. The top 10 cm was replaced with fresh field
soil before planting and all microcosms were irrigated with tap water.
The microcosms were destructively sampled at
growth stage (GS) [12,13] 24 (tillering), 69
(flowering) and 92 (ripening). At each of the
sampling occasions, the PVC casing of twelve
microcosms (three treatments, four replicates)
were cut lengthwise and removed without disturbing the soil. The roots from the top 15 cm and the
layer 30-60 cm of each soil core were taken for
microbial analyses. The roots were cleaned by
placing the soil and roots taken from each microcosm in a bucket of tap water for approx. 3 h to
loosen the adhering soil. Most soil was then carefully removed by hand. The remaining loose soil
was removed with two rinses in clean tap water
before roots were blotted dry, cut into small
252
segments (approx. 2 cm length) and mixed thoroughly by hand. From each of these root samples,
a 1 g (fresh weight) sub-sample was taken and
crushed in 9 ml RA with a pestle and mortar.
Ten-fold dilutions were prepared in RA. The
dilution series prepared from the roots taken
from the top 15 cm of soil were plated onto 0.1
strength TSA for the enumeration of culturable
bacterium and actinomycete propagules, Pseudomonas selective medium (P-l) [10] to enumerate fluorescent Pseudomonas propagules, P-1
medium amended with 100 ppm kanamycin and
50 ppm X-gal (P-1KX) [3] for the enumeration of
recombinant P. aureofaciens propagules, Potato
Dextrose Agar (Oxoid) amended with 100 ppm
streptomycin sulphate and 50 ppm rose bengal
(PDA ÷) for the enumeration of filamentous fungi
and yeast propagules and Modified Mircetich
medium (VP3) [14] for the enumeration of
Pythium propagules. Plates were incubated at
25°C and the different microbial components were
enumerated at 6x magnification. Incubation times
for the different media were 2-3 days for VP3, 6
days for P-1 and P-1KX and 10 days for 0.1
strength TSA and PDA +.
At GS 69 (flowering) and 92 (ripening) the
microbial populations on roots extracted from the
top 15 cm and the layer 30-60 cm, were plated
onto P-1 medium and incubated at 25°C for up to
10 days. Colonies were enumerated on a daily
basis for six consecutive days and at day 10. In
this way, seven counts (or classes) were generated
per plate, i.e. colonies that were visible after one,
two, three, four, five, six and ten days. Plates that
contained between 5 and 200 colonies were selected for enumeration. When plates became too
crowded, the next dilutions were used for enumeration. The number of bacteria in each class
was expressed as a proportion (%) of the total
number of culturable bacteria. The distribution
of classes in the different samples could then be
compared using multinomial analyses [15].
Effect of P. aureofaciens seed inoculations on wheat
seedlings
Shoot weight as well as emergence was greater
(P < 0.05) when wheat seeds were inoculated with
recombinant P. aureofaciens. Root weight was
not significantly affected by the bacterial inoculum (Table 1).
Six days after planting, seed inoculation with
P. aureofaciens caused a 98% reduction (P < 0.01)
of the indigenous Pseudomonas population o n
seed (Table 2). Total Pseudomonas populations
(indigenous + recombinant) were not significantly
affected by the seed inoculations with P. aureofaciens (Table 2).
Effect of P. aureofaciens spray application at tillering on microbial populations on the phylloplane at
booting (GS 47)
Eight pots, each containing 10 wheat plants at
GS 24 (tillering) were sprayed until run-off with a
Effect of P. aureofaciens seed inoculations on microbial populations on roots at tillering, flowering
and ripening
Total bacterial counts on 0.1 strength TSA
were not significantly affected by the different
suspension of 2.3 × 109 recombinant P. aureofaciens cells ml-1 0.25 strength Ringer's solution.
The bacterial suspension was amended with
0.01% (v/v) Tween 80 to improve contact of the
suspension with the leaf surface. Eight control
pots were sprayed with a 0.01% Tween 80 solution without recombinants. After 28 days (booting, GS 47), leaves 1, 3, 5 and the flag leaves were
harvested separately and populations of culturable bacteria, Pseudomonas, filamentous fungi,
white and pink yeasts were extracted by macerating each weighed sample in a blender for 2 min in
50 ml, 0.25 strength Ringer's solution. A dilution
series was prepared, and the different microbial
components were quantified as described before
except fungal propagules were quantified on PDA
amended with 20 ppm chlortetracyclin hydrochloride.
Statistical analyses
Unless otherwise stated, results were analysed
using analyses of variance. To normalise data
sets, data were either log10 transformed or logit
transformed, if expressed as a percentage, before
they were analysed and compared using an F-test.
Results
253
treatments, but populations increased (P < 0.001)
as the plants matured from around l0 s cfu (g
fresh wt. root) -1 at GS 24 to almost 109 cfu (g
fresh wt. root) -1 at GS 92 (Table 3). Actinomycetes were not significantly affected by the
different treatments (Table 3). As plants matured, actinomycete populations increased (P <
0.001) from 1.1 × 107 cfu (g fresh wt. root) -1 at
GS 24 to 5.6 × 107 cfu (g fresh wt. root)-I at GS
92 (Table 3). Total Pseudomonas populations on
P-1 medium were not significantly affected by the
different treatments, but increased (P < 0.001)
from 1.6 × 107 cfu (g fresh wt. root)- 1 at GS 24 to
4.1 x 107 cfu (g fresh wt. root) -x at GS 92 (Table
3). Recombinant populations on the roots remained around 105 cfu (g fresh wt. root) -1 from
GS 24 to GS 92 (Table 3). Filamentous fungal
counts on PDA + were not significantly affected
by the different treatments (Table 3). In time,
filamentous fungal counts increased (P < 0.001)
from 2.2 × 105 cfu (g fresh wt. root)-1 at GS 24,
to 9.0 × 105 cfu (g fresh wt. root) -~ at GS 92
(Table 3). Total yeast counts on PDA ÷ on the
roots were not significantly affected by plant age
or treatment, and stayed at a level of around
3.0 × 104 cfu (g fresh wt. root) ~1 from GS 24 to
Table 1
Effect of seed inoculation with Pseudomonas aureofaciens
SBW25EeZY-6KX on root weights, shoot weights and emergence, 5 d after sowing. A control (C) treatment was compared with two bacterial seed treatments (I); n ~ 4
Treatment
Root
weight
(g fresh
weight)
Shoot
weight
(g f r e s h
weight)
Emergence
(from 25
seeds)
C + guar gel a
I b _ guar gel
I + guar gel
S.E.D. c (df d = 6)
Fprob e
0.15
0.20
0.19.
0.021
NS
0.37
0.44
0.44
0.020
*
13.8
22.5
20.0
2.14
*
a Seeds planted in 1.25% guar gel.
b Seeds inoculated by vacuum infiltration to give 107 bacterial
cells per seed.
c S.E.D. = Standard Error of the Difference between two
means.
d df = degrees of freedom.
probability levels from analysis of variance: NS = not significant; * = P < 0.05; ** = P < 0.01; * * * = P < 0.001.
Table 2
Effect of seed inoculations with Pseudomonas aureoj:aciens
SBW25EeZY-6KX on indigenous Pseudomonas populations
on seeds, roots and leaves 6 d after sowing (GS 11). A control
(C) treatment was compared with two bacterial seed treatments (I). Data are expressed as logl0(cfu per plant tissue
type + 1); n = 4
Site
Treatment
Indigenous
Pseudomonas
Seeds:
C + guar gel a
I b _ guar gel
I + guar gel
Roots
C + guar gel
I - guar gel
I + guar gel
Leaves C + guar gel
I - guar gel
I + guar gel
S.E.D. e (dr) d
F probe
between treatments
between sites
interaction
Recombinants
Total
Pseudomonas
6.89
5.28
4.91
5.31
4.85
5.04
3.66
3.35
3.13
0.234 (18)
0.208 (6)
6.89
7.55
7.55
7.46
7.46
5.31
5.42
5.54
5.24
5.46
3.66
4.45
4.52
4.23
4.28
0.225 (12) 0.254 (18)
0.178 (3)
0.162 (6)
**
***
***
NS
***
NS
NS
***
NS
a Seeds planted in 1.25% guar gel.
b Seeds inoculated by vacuum infiltration to give 8.2 + 1.1 × 106
bacterial cells/seed.
c S.E.D. = Standard Error of the Difference. The first S.E.D.
is for comparison between any set of means, the second for
comparisons between sites. Each replicate consisted of five
plants.
d df = degrees of freedom.
e probability levels from analysis of variance: NS = not significant; * = P < 0.05; * * = P < 0.01; * * * = P < 0.001.
GS 92 (Table 3). Pythium counts on VP3 medium
were not significantly affected by the different
treatments, but increased ( P < 0.01) from 8.4 ×
102 cfu (g fresh wt. root) -1 at GS 24, to 2.0 × 103
cfu (g fresh wt. root)-1 at GS 92 (Table 3).
Although statistically significant interactions
were observed for many of the variables, these
were almost invariably linked to non-significant
effects of treatments when averaged across growth
stages. There appeared to be very little pattern to
the variation within growth stages and so no
biological significance was attached to them.
At GS 69 Pseudomonas population structures
on roots that were not inoculated with P. aureofaciens were dominated ( > 60%) by Pseu-
254
Table 3
Effect of Pseudomonas aureofaciens (wild-type SBW25 and recombinant SBW25EeZY-6KX) applied as a seed coating of 5 × 108
cfu/seed on different components of the microbial populations on the roots of spring wheat at GS 24 (tillering), 69 (flowering) and
92 (ripening). Data are expressed as logt0 cfu (g fresh wt. root)-t; n = 4
Growth
stage a
Tillering
(GS 24)
Treatment
Control
Wild-type
Recombinant
Flowering
Control
(GS 69)
Wild-type
Recombinant
Ripening
Control
(GS 92)
Wild-type
Recombinant
S.E.D. b (dr) c
F prob d
between treatments
between growth stages
interaction
a
b
c
d
Total
culturable
bacteria
Actinomycetes
Pseudomonas
Recombinant
Filamentous
fungi
Yeasts
Pythium
8.07
7.82
8.13
8.45
8.72
8.49
8.87
8.75
8.80
0.061 (27)
7.32
6.77
6.83
7.30
7.44
7.57
7.51
7.86
7.81
0.178(27)
7.17
6.80
7.43
6.90
7.21
6.95
7.69
7.62
7.49
0.219(27)
5.85
4.69
5.06
0.429(9)
5.58
5.25
5.06
5.19
5.61
5.55
6.12
5.93
5.72
0.163 (27)
4.51
4.23
4.33
4.07
4.51
4.59
4.55
4.25
4.57
0.163 (27)
2.99
2.96
2.79
3.00
3.16
3.10
3.29
3.20
3.41
0.207(27)
NS
***
***
NS
***
**
NS
***
*
NS
NS
***
**
NS
NS
**
NS
**
NS
Growth stage index system for cereals according to Zadoks et al. [13].
S.E.D. = Standard Error of the Difference; S.E.D. is for comparison between any set of means.
df = degrees of freedom.
probability levels from analysis of variance: NS = not significant; * = P < 0.05; * * = P < 0.01; * * * = P < 0.001.
domonas spp. t h a t d e v e l o p e d visible c o l o n i e s o n
P-1 m e d i u m , 2 days a f t e r i n o c u l a t i o n . W h e n roots
f r o m t h e top 15 c m o f t h e m i c r o c o s m s w h i c h w e r e
t r e a t e d with r e c o m b i n a n t or wild-type P. aureofaciens w e r e p l a t e d o n P-1 m e d i u m , m o s t c o l o n i e s
( > 3 5 % ) w e r e visible only a f t e r 3 days i n c u b a t i o n
(Fig. 1). T h e s e d i f f e r e n c e s in p o p u l a t i o n structures b e t w e e n t h e u n t r e a t e d c o n t r o l a n d t r e a t m e n t s with e i t h e r r e c o m b i n a n t or wild-type P.
aureofaciens w e r e highly significant ( P < 0.001).
N o such d i f f e r e n c e s in Pseudomonas p o p u l a t i o n
s t r u c t u r e s w e r e f o u n d on th e r o o ts in th e layers
3 0 - 6 0 cm b e t w e e n c o n t r o l a n d wild-type treatm e n t s (Fig. 1). T h e level o f r o o t c o l o n i s a t i o n by
t he i n t r o d u c e d Pseudomonas was very low on
t h e s e roots ( < 100 cfu (g fresh wt. r o o t ) - l ) . A t
G S 92 t h e r e w e r e no significant d i f f e r e n c e s b e t w e e n t h e p o p u l a t i o n s t r u c t u r e s o f Pseudomonas
p o p u l a t i o n s e x t r a c t e d f r o m roots at d i f f e r e n t
d e p t h s (Fig. 2).
Effect of P. aureofaciens spray application at tillering on microbial populations on the phylloplane at
booting
T w e n t y - e i g h t days a f te r spray a p p l i c a t i o n
(booting, G S 47) with r e c o m b i n a n t s , i n d i g e n o u s
m i cr o b i al p o p u l a t i o n s that i n h a b i t e d t h e phyllop l a n e o f w h e a t w e r e n o t significantly a f f e c t e d by
t h e p r e s e n c e o f t h e r e c o m b i n a n t ( T a b l e 4). A l t h o u g h no overall effect on t h e i n d i g e n o u s microbial p o p u l a t i o n s was found, a significant effect
( P < 0.01) on t h e i n d i g e n o u s Pseudomonas p o p u lations o n l e a f 1 was o b s e r v e d as a result of a
spray a p p l i c a t i o n with r e c o m b i n a n t s ( T a b l e 4).
T h e effect of t h e r e c o m b i n a n t on p i n k yeasts on
t h e flag l e a f can be a t t r i b u t e d to t h e failure to
d e t e c t p i n k yeasts on t h e c o n t r o l t r e a t m e n t and is
t h e r e f o r e r e g a r d e d as biologically insignificant
( T a b l e 4).
A l l m i c r o b i a l p o p u l a t i o n s e n u m e r a t e d on t h e
leaves i n c r e a s e d ( P < 0.001 for total c u l t u r a b l e
bacteria, Pseudomonas, pink yeasts, f i l a m e n t o u s
fungi an d Cladosporium; P < 0 . 0 5
for w h i t e
yeasts) with l e a f age ( T a b l e 4).
Discussion
Ef f ect s o f s e e d i n o c u l a t i o n s with P. aureofaciens o n w h e a t plants a n d t h e m i c r o b i a l p o p u l a tions associated with the p h y t o s p h e r e o f t h o s e
255
0-15cm
plants were most pronounced at the seedling
stage (GS 11). Seed inoculations with recombinant P. aureofaciens resulted in a 98% reduction
60
"I
50
0-15cm
60
40
50
30
40
20
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Fig. 1. Percentages of total Pseudomonas colony counts as
they appeared on Pseudomonas selective agar (P-l) over a
period of 10 days. Populations were extracted at growth stage
69 (flowering) from wheat roots taken from undisturbed soil
microcosms that were placed in the glasshouse. Roots were
taken from depths of 0-15 cm and 30-60 cm. Seeds were
treated with sterile methyl cellulose (MC) (o), M C + 5 × 10 s
wild-type P. aureofaciens per seed ( I ) and M C + 5 x l 0 8
recombinant P. aureofaciens per seed ( • ) ; n = 4.
Fig. 2. Percentages of total Pseudomonas colony counts as
they appeared on Pseudomonas selective agar (P-l) over a
period of 6 days. Populations were extracted at growth stage
92 (ripening) from wheat roots taken from undisturbed soil
microcosms that were placed in the glasshouse. Roots were
taken from depths of 0-15 cm and 30-60 cm. Seeds were
treated with sterile methyl cellulose (MC) (o), MC+ 5 × 108
wild-type P. aureofaciens per seed (It) and MC+SX108
recombinant P. aureofaciens per seed ( • ); n = 4.
o f t h e i n d i g e n o u s Pseudomonas p o p u l a t i o n s o n
wheat seeds and more than 50% reduction of
indigenous
Pseudomonas
populations
on
roots
256
and leaves 5 d after planting. Although this strain
of Pseudomonas should not be regarded as a
biological control agent, the large bacterial load
on the seed and the subsequent colonisation of
the roots may have prevented the invasion of
detrimental soil microorganisms onto the seeds
and roots [16] and could explain the improved
emergence and increased shoot weight of those
seedlings treated with P. aureofaciens.
Even though the introduced P. aureofaciens
survived on the roots of wheat in the top 15 cm of
soil until maturity in excess of 105 cfu (g flesh wt.
root) -t, effects on the total culturable populations of indigenous microbial populations were
not significant, and the presence of the introduced bacterium did not seem to interfere with
the natural microbial population development on
maturing roots. Different results were obtained
when the Pseudomonas community structures of
the different treatments were compared. At GS
69 (flowering) but not at GS 92 (ripening), indigenous populations of Pseudomonas on roots in the
top layer (0-15 cm) of the microcosms that were
treated with wild-type or recombinant P. aureola-
ciens were significantly different from the control
treatment in that P. aureofaciens-treated roots
had a population that was dominated by isolates
that developed visible colonies on P-1 medium
after 3 days, whilst the control treatment was
dominated by Pseudomonas isolates that developed visible colonies on P-1 medium after 2 days.
A shift towards more slowly growing Pseudomonas isolates was only found on roots in the
top 15 cm of soil that were colonised by a relatively large population of introduced P. aureofaciens, but such a shift was not found on roots
taken from the same root systems in the lower
layer (30-60 cm) that were colonised by a relatively small population of the introduced Pseudomonas. This suggests thai this community shift
in the top layers was a result of direct competition between the introduced Pseudomonas populations and the indigenous ones, and that it was
not the result of sampling error or changes in
plant physiology.
Culturable microbial populations on the phylloplane were not significantly affected by the
introduction of a large inoculum of P. aureofa-
Table 4
E f f e c t o f r e c o m b i n a n t Pseudomonas aureofaciens S B W 2 5 E e Z Y - 6 K X o n i n d i g e n o u s m i c r o b i a l p o p u l a t i o n s o n t h e p h y l l o p l a n e o f
w h e a t ( l e a f 1, 3, 5 a n d f l a g leaf), 28 d a f t e r a s p r a y a p p l i c a t i o n a t G S 24 w i t h 108 b a c t e r i a l cells m l -~. D a t a a r e e x p r e s s e d as log10
cfu (g f r e s h wt. r o o t ) - 1 ; n = 4
Total
culturable
bacteria
Pseudomonas
White
Yeasts
Pink
Yeasts
Filamentous
fungi
Cladosporiurn
Leaf 1
Control
Recombinant
Leaf 3
Control
Recombinant
Leaf 5
Control
Recombinant
Flag leaf
Control
Recombinant
S . E . D . a ( d f d = 36)
( d f = 12)
F probc
9.17
9.15
8.08
8.36
6.88
7.13
5.21
5.39
0.142
0.131
5.26
3.61
6.27
6.08
5.68
6.07
0.607
0.544
4.17
3.45
4.26
4.24
2.68
2.14
1.04
2.54
1.121
1.212
7.04
6.89
4.82
5.72
5.27
5.41
4.41
0.404
0.400
7.71
7.79
6.06
6.09
5.17
5.10
4.35
4.66
0.149
0,144
7.41
7.64
5.58
5.71
4.63
3.45
1.61
0.43
0.656
0.691
between treatments
b e t w e e n sites
interaction
NS
***
NS
NS
***
**
NS
*
NS
NS
***
***
NS
***
NS
NS
***
NS
Site
Treatment
Indigenous
a S . E . D . = S t a n d a r d E r r o r o f t h e D i f f e r e n c e b e t w e e n t w o m e a n s ; t h e first S . E . D . is f o r c o m p a r i s o n b e t w e e n a n y set o f m e a n s , t h e
second for comparisons between treatments.
b df = degrees of freedom.
c p r o b a b i l i t y levels f r o m a n a l y s i s o f v a r i a n c e : N S = n o t s i g n i f i c a n t ; * = P < 0.05; * * = P < 0.01; * * * = P < 0.001.
257
ciens. This suggests that either the enumeration
of total culturable populations of bacteria and
fungi is not sensitive enough to pick up subtle
differences, or that most propagules that are on
the leaf surface are in a metabolically inactive
stage (conidia, resting spores, etc.) and thus unable to respond to the introduction of the bacterial inoculum.
From the results in this paper, it is clear that
the introduction of innocuous microorganisms
onto the wheat seeds can cause perturbations of
the natural microbial populations in the phytosphere. Even though, as a result of seed inoculation, some of these perturbations exceeded 2 log
units, there was (in this case) no reason to perceive them as a risk to the environment, as these
effects only persisted for a short time. Medium
and longer term effects of seed inocula with P.
aureofaciens never exceeded microbial perturbations in excess of 0.5 of a log unit. Therefore, if
this or a functional GEMMO were to be introduced into the environment, perturbations of
non-target microbial populations of the kind
found here should be acceptable and would perhaps need to be several magnitudes greater to
perceive them as a risk to the environment.
Acknowledgements
This work was carried out under contract
PECD 7-8-161 and PECD 7-8-143 of the Department of the Environment's Genetically Modified
Organisms Research Programme. The authors
wish to thank Dr. M.J. Bailey for providing us
with the chromosomally marked Pseudomonas
strain. The views expressed in this paper are
those of the authors and not necessary of the
Department of the Environment.
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