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A Pseudomonas fluorescens F113 mutant with enhanced competitive 1
colonization ability shows improved biocontrol activity against fungal 2
root pathogens 3
4
Emma Barahona, 1†
Ana Navazo, 1†
Francisco Martínez-Granero, 1
Teresa Zea-5
Bonilla, 2
Rosa María Pérez-Jiménez, 2
Marta Martín, 1
and Rafael Rivilla. 1*
6 7
1Departamento de Biología, Universidad Autónoma de Madrid, 28049 Madrid, Spain. 8 2
Instituto de Investigación y Formación Agraria y Pesquera, Centro de Churriana, 9
29140 Málaga. Spain. 10
11
12
13
14
Running Title: Competitive rhizosphere colonization and biocontrol 15
16
17
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19
*Corresponding author: Rafael Rivilla. 20
21
23
24
† Both authors contributed equally 25
26
27
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00320-11 AEM Accepts, published online ahead of print on 17 June 2011
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Motility is one of the most important traits for efficient rhizosphere colonization 1
by Pseudomonas fluorescens F113. In this bacterium, motility is a polygenic trait 2
that is repressed by at least three independent pathways, including the Gac 3
posttranscriptional system, the Wsp chemotaxis-like pathway and the SadB 4
pathway. Here we show that the kinB gene which encodes a signal transduction 5
protein that together with AlgB has been implicated in alginate production, 6
participates in swimming motility repression through the Gac pathway acting 7
downstream of the GacAS two-component system. Gac mutants are impaired in 8
secondary metabolites production and are unsuitable as biocontrol agents. 9
However, the kinB mutant and a triple mutant affected in kinB, sadB and wspR 10
(KSW) possess a wild-type phenotype for secondary metabolism. The KSW strain 11
is hypermotile and more competitive for rhizosphere colonization than the wild-12
type strain. We have compared the biocontrol activity of KSW with the wild-type 13
strain and a phenotypic variant (V35) which is hypermotile and hypercompetitive 14
but is affected in secondary metabolism since it harbors a gacS mutation. 15
Biocontrol experiments in the Fusarium oxysporum f.sp. radicis-lycopersici (FOL)/ 16
Lycopersicum esculentum (tomato) and Phytophthora cactorum (Pc)/ Fragaria vesca 17
(strawberry) pathosystems have shown that the three strains possess biocontrol 18
activity. Biocontrol activity was consistently lower for V35, indicating that the 19
production of secondary metabolites was the most important trait for biocontrol. 20
Strain KSW showed improved biocontrol when compared with the wild-type 21
strain, indicating that an increase in competitive colonization ability resulted in 22
improved biocontrol and that the rational design of biocontrol agents by mutation 23
is feasible. 24
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Pseudomonas fluorescens F113 is a biocontrol agent isolated from the sugar-beet 1
rhizosphere (36) that is able to suppress take-all disease produced by the oomycete 2
Pythium ultimum (17). This strain, which is able to efficiently colonize the rhizosphere 3
of a variety of plants (2, 14, 26, 32) is being used as a model for rhizosphere 4
colonization (5, 25, 32) and has also been genetically modified for polychlorinated 5
biphenyls (PCBs) rhizoremediation (3, 7, 40).. The biocontrol ability of this strain has 6
been related to the production of a series of secondary metabolites, including 7
siderophores, di-acetyl-phloroglucinol (DAPG), hydrogen cyanide and an extracellular 8
protease (1, 35), whose production is regulated by the GacAS post-transcriptional 9
system (1). 10
The GacAS system, as well as SadB and the Wsp system, independently regulates 11
swimming motility in strain F113 (28). In this strain mutants affected in sadB and wspR 12
showed increased swimming motility. In F113, the sadB and wspR genes showed 84% 13
and 83% identity with their P. aeruginosa orthologues and are highly conserved in 14
sequence and genomic context with orthologues in all the sequenced P. fluorescens 15
strains. SadB and WspR possibly repress swimming motility through the secondary 16
messenger c-di-GMP, since WspR possesses a diguanilate cyclase activity (20) and 17
SadB contains a modified HD domain, typical of c-di-GMP phosphodiesterases (8). We 18
have previously shown that a triple mutant gacSsadBwspR (GSW) is hypermotile (28) 19
and is more competitive for rhizosphere colonization than the wild-type strain, 20
displacing it from the rhizosphere (5). We have also shown that hypermotile phenotypic 21
variants are selected by the rhizospheric environment (25, 32). Some of these variants, 22
such as variant 35 (V35), that harbors a gacS mutation and other unidentified mutations 23
causing hypermotility, are also more competitive than the wild-type strain for 24
rhizosphere colonization (25). 25
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The ability to colonize the rhizosphere has been considered a relevant trait for 1
biocontrol. Kamilova et al., (21) showed that a Collimonas fungivorans strain that 2
colonized the tomato rhizosphere was able to control the plant-pathogenic fungus 3
Fusarium oxysporum f.sp. radicis-lycopersici by occupying the same sites in the root 4
that the fungus and competing for nutrients. Furthermore, Pliego et al., (30) showed that 5
two rhizosphere-colonizing pseudomonads had different biocontrol abilities over 6
Rosellinia necatrix on avocado roots. The higher biocontrol ability of one of the strains 7
was attributed to its colonization strategy, and it was considered that the sites occupied 8
by the bacteria were as important as the overall colonization level. 9
In the case of P. fluorescens F113 derivatives, hypercompetitivity of the GSW and V35 10
strains was linked to hypermotility and no differences with the wild-type strain were 11
observed for colonization level or pattern (5). However, these highly competitive strains 12
are not suitable for biocontrol, since they harbor mutations in the GacAS system and are 13
therefore unable to produce secondary metabolites. 14
Here we identify a mutation in the kinB gene, which encodes the sensor protein of a two 15
components system (KinB/AlgB) that also results in hypermotility. Although this gene 16
appears to act in the GacAS pathway, it does it downstream gacA and gacS and 17
therefore presents a wild-type phenotype for secondary metabolism. The aim of this 18
work is to test whether an increase in competitive rhizosphere colonization ability is 19
linked to a better performance as a biocontrol agent. 20
21
MATERIALS AND METHODS 22
Microorganisms, growth conditions and plasmids. Strains and plasmids used are 23
listed in Table I. P. fluorescens strains were grown in SA medium (34) with shaking 24
overnight at 28oC. 1.5% purified agar (Pronadisa, Spain) was added for solid medium. 25
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Pseudomonas strains were grown in LB medium for biocontrol assays. Escherichia coli 1
strains were grown in LB medium with shaking overnight at 37oC. When required, the 2
following antibiotics were added: rifampicin 100 µg/ml (for P. fluorescens), 3
spectinomycin 100 µg/ml (for P. fluorescens), tetracycline 10 µg/ml (for Escherichia 4
coli) or 70 µg/ml (for P. fluorescens), and kanamycin 25 µg/ml (for E. coli) or 50 µg/ml 5
(for P. fluorescens). 6
Fusarium oxysporum f sp. radicis-lycopersici was stock cultured on potato dextrose 7
agar (PDA) and grown in Czapek-Dox liquid medium (37) at 25°C. Media were 8
solidified with 1.8% agar, when necessary. 9
Phytophthora cactorum was grown in Oatmeal Agar (OMA) medium during 10 days at 10
25oC and then was stock cultured on PDA for 11 days at 25
oC. 11
12
Physiological and enzymatic assays. Swimming was tested on SA medium with 0.3% 13
purified agar. Bacteria were inoculated with a sterile toothpick and incubated at 28oC. 14
Swimming haloes were measured after 18 hours. Exoprotease production was tested on 15
skimmed milk medium (24) by the appearance of a degradation halo after 24 hours. 16
Pyoverdine production under iron-sufficient conditions was observed by exposing 17
cultures grown on LB plates to U.V. light as previously described (24). Hydrogen 18
cyanide (HCN) production was detected using the method described by Castric and 19
Castric (10). Whatman 3MM chromatography paper was impregnated by a solution 20
composed by copper(II) ethyl acetoacetate (5 mg/mL) and 4,4'-methylenebis-(N,N-21
dimethylaniline) (5 mg/mL) in chloroform and were dried in darkness. The potentially 22
cyanogenic organisms were grown on individual agar plates containing SA media, with 23
the HCN detection paper on the lid of the plates, and incubated one day at 28ºC in 24
darkness. Paper changes from white to dark blue color indicate a positive HCN reaction. 25
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Generation and analysis of mutants. Directed mutagenesis was performed by cloning 2
an internal fragment of the target gene into the suicide vectors pK18mobsacB (32) or 3
pG18mob2 (22). The constructs were introduced into F113 or F113-derived strains by 4
triparental mating using pRK600 (19) or pRK2013 (18) as helper plasmids and selected 5
for homologous recombination. The same methods were used in the construction of 6
double and triple mutants. All mutants were checked by Southern-Blot and PCR. 7
8
Rhizosphere competitive colonization assays. Alfalfa seeds (var. resis) were surface 9
sterilized in 70% ethanol for 2 min and then in diluted bleach (1:5) (final sodium 10
hypochlorite concentration 1%) for 15 min and rinsed thoroughly with sterile distilled 11
water. Seed vernalization was performed at 4oC for 16 h followed by incubation in 12
darkness at 28oC for 1 day for germination. Germinated alfalfa seeds were sown in 13
Leonard jar gnotobiotic systems (39) using ca. 3.5 L sterile perlite as the solid substrate 14
and 8 mM KNO3- supplemented FP (16) as the mineral solution (500 ml/jar; replenished 15
every two days). After 2 days, alfalfa seeds were inoculated with ca. 108 cells of the 16
appropriate strains prepared by culture dilution (108 cells/ml = 0.0138 OD600). In 17
competition experiments, strains were inoculated at a 1:1 ratio. In all cases 1ml diluted 18
culture was inoculated per plant. Plants were maintained under controlled conditions for 19
2 weeks: 16 h in the light (100 µmol/m2/s) at 25
oC and 8 h in the dark at 18
oC; 60-70% 20
humidity. Bacteria were recovered from the rhizosphere by vortexing the roots tips (last 21
centimeter of the main root) for 2 min in a tube containing FP medium and plating the 22
appropriate dilutions on SA plates supplemented with appropriated antibiotics. Every 23
experiment was performed three times with three replicates each time, and every 24
replicate contained at least 20 plants. 25
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Biocontrol assays 2
Fusarium oxysporum f sp. radicis-lycopersici (FOL)/ Lycopersicum esculentum 3
(tomato) system. 4
Tomato (var. juliana) seeds were surface sterilized in 70% ethanol for 2 min and then in 5
diluted bleach (1:5) for 15 min and rinsed thoroughly with sterile distilled water. Seeds 6
were coated with bacteria by incubation for 15 minutes in a LB grown overnight 7
culture. This method yielded a seed coating of ca. 7x105
cfu /seed. Inoculated seeds 8
were planted in pots containing sterile perlite: peat-based commercial substrate (50:50) 9
mixed thoroughly with 3x106 spores/L when necessary. Tomato plants were maintained 10
under controlled conditions (18h in the light at 24oC and 8 h in the dark at 16
oC) for 3 11
weeks and were supplied with sterile distilled water four times/week. Fifty plants were 12
used for each treatment and each control, including controls with no microorganisms). 13
Dead and symptomatic plants were determined after 21 days. Roots were removed, cut 14
up into pieces, surface sterilized and were incubated on PDA-acid medium at 25oC to 15
determinate pathogen recovery. All data were compared using statistic application 16
software SPSS. Statistical significance was calculated by the Bonferroni's test of variance 17
analysis (ANOVA, p<0.05). 18
19
Phytophthora cactorum (Pc)/ Fragaria vesca (strawberry) system. 20
Strawberry (var. camarosa) clonal plants (micropropagated) were grown in sterile jars 21
and then transplanted in 200 ml pots with sterile soil: sand: perlite (11:4:2) and watered 22
with sterile water two times/week. After plant acclimatization for 4 weeks (twenty 23
plants for each treatment and each control), surface sterilized roots (diluted bleach, final 24
hypochlorite concentration 0.06%, 20 min) were coated during 20 minutes with each 25
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strain previously grown in LB medium (OD600 0.9) and washed in phosphate buffer (pH 1
7.2, 0.1M). Phytophthora cactorum zoospores were obtained by a heat shock: 2h at 2
10oC and then 1h at room temperature to release zoospores from sporangium. A 1:1:1:1 3
mixture of four Phytophthora cactorum strains was used (Table I). Plants were 4
transplanted in pots again and after two days the pathogen suspension (5x104 zoospores/ 5
L) was added by watering. Dead and symptomatic plants were determined 15 days later. 6
Roots were removed, cut up into pieces, surface sterilized and were incubated on 7
P5ARP medium (15) at 25oC to determinate pathogen recovery. All data were compared 8
using statistic application software SPSS. Statistical significance was calculated by the 9
Bonferroni's test of variance analysis (ANOVA, p<0.05). 10
11
12
13
14
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RESULTS 16
Phenotypic analysis of kinB mutants. During the screening of a Pseudomonas 17
fluorescens F113 mutant library for hypermotile mutants we found a mutant harboring a 18
transposon insertion within the kinB gene that was more motile than the wild-type strain 19
(28). Transposon insertion was located within a gene showing 86% sequence identity 20
with the P. fluorescens kinB gene. Like in Pf01 and other pseudomonads, this gene is 21
the last gene in a bicistronic operon and is preceded by the algB gene (86% sequence 22
identity with the Pf01 orthologue). In order to test that the hypermotile phenotype was 23
due to the disruption of the kinB gene, the mutant was reconstructed by the insertion 24
through homologous recombination of a suicide plasmid containing an internal 25
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fragment of kinB that interrupted the gene, as checked by Southern-blot. This mutant 1
was used for further analysis. A similar mutant was constructed by disruption of the 2
algB gene, encoding its cognate response regulator. As shown in Fig. 1, the kinB mutant 3
showed increased swimming motility when compared to the wild-type strain. The algB 4
mutant did not show differences in swimming motility with the wild-type strain, 5
indicating that the hypermotile phenotype was caused by the lack of a functional kinB 6
gene. Polar effects of the mutations can be disregarded since the mutation in the first 7
gene of the operon, algB has no swimming phenotype and the gene downstream of kinB 8
is transcribed in the opposite direction. Furthermore introduction of plasmid pBG1708, 9
which contains the cloned algB and kinB under the control of its own promoter had no 10
effect on the algB mutant and restored normal swimming phenotype to the kinB mutant, 11
indicating a the genetic linkage of the swimming phenotype with the kinB mutation. 12
None of these mutants showed altered mucoidy. 13
We have previously shown that at least three independent pathways repress motility in 14
P. fluorescens (28). In order to test whether kinB is related to any of these pathways, 15
double mutants affecting kinB and each of the gacS, sadB and wspR genes, representing 16
the three different pathways, were constructed. As shown in Fig. 1, the double mutants 17
kinBsadB (KS) and kinBwspR (KW) showed an additive swimming phenotype, 18
indicating that kinB participates in a pathway different to the SadB or WspR pathways. 19
Conversely, the double mutant kinBgacS (KG) presented a swimming phenotype 20
identical to the gacS mutant. Therefore gacS is epistatic over kinB indicating that both 21
genes could act in a same pathway. Since gacS mutants are affected in secondary 22
metabolism, lacking exoprotease secretion and overproducing the siderophore 23
pyoverdine under iron-sufficient conditions (24), we tested these traits in the kinB and 24
kinBgacS mutants. As shown in Fig. 2, only the kinB mutant presented a wild-type 25
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phenotype with normal production of exoprotease and pyoverdine, while the kinBgacS 1
showed a gacS phenotype for both characters. Taken together, these results show that 2
gacS acts upstream of kinB in repressing swimming motility. 3
We also constructed a triple mutant kinBsadBwspR (KSW). The triple mutant showed 4
an additive swimming phenotype with a motility that was equivalent to the motility of a 5
gacSsadBwspR (GSW) mutant (27) and also to the motility of the rhizosphere selected 6
variant 35 (V35) (28) (Fig. 1). Like these two strains, the KSW mutant was defective in 7
biofilm formation on abiotic surfaces. Since both GSW and V35 are more competitive 8
for rhizosphere colonization than the wild type strain (25), we also tested the 9
competitive colonization ability of the KSW mutant by using an alfalfa root-tip assay 10
(9). As shown in Fig.3, the KSW mutant is more competitive than the wild-type strain, 11
being able to displace the wild-type strain from the root tip. Furthermore, KSW presents 12
a wild-type phenotype for exoprotease, pyoverdine (Fig. 2) and hydrogen cyanide 13
production. Since the KSW mutant is not affected in secondary metabolism it is likely 14
to be more suitable for biocontrol, we tested the hypothesis that KSW higher 15
competitivity results in a better performance in biocontrol than the wild-type strain. For 16
these experiments we have compared the KSW mutant with the wild-type strain and 17
with V35, a hypercompetitive strain but impaired in secondary metabolism, since it 18
harbors a gacS mutation (25). 19
20
Analysis of biocontrol on tomato. The first model used was tomato plants infected 21
with the pathogenic fungi Fusarium oxysporum f.sp. radicis-lycopersici (FOL). As 22
shown in Fig. 4, infection with the fungi resulted on severe effects on the tomato plants: 23
more than 20% of the plants were dead after 21 days (Fig. 4A) and 83% were either 24
dead or showed diverse disease symptoms. These results indicate a high degree of 25
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infection. 27% of the surviving plants showed symptoms on aerial parts consisting in 1
chlorotic leaves with necrotic spots (Fig. 4B). Even more effects were observed in the 2
root, with over 75% of the surviving plants showing necrosis (Fig. 4C). Furthermore, 3
we re-isolated the fungi from 37% of the roots (Fig. 4D). Inoculation with any of the 4
bacterial strains resulted in a decrease in infection: dead plus symptomatic plants values 5
were 44% for inoculation with the wild-type, 49% for inoculation with V35 and 33% 6
for inoculation with KSW. This effect was observed for all the parameters tested, 7
indicating that all the strains possessed biocontrol ability (Fig. 4). For all the parameters 8
we observed the same pattern, with V35 as the strain with less biocontrol ability. 9
Although the average values for all parameters were lower in the case of inoculation 10
with V35 when compared with non inoculated plants, statistically significant differences 11
(p<0.05) were only observed for root necrosis (Fig. 4C). Conversely, a clear biocontrol 12
effect was observed when infected plants were inoculated with either the wild-type 13
strain F113 or with the KSW mutant. Differences were statistically significant (p<0.05) 14
for all the parameters except for the recovery of the pathogen from the root in the case 15
of inoculation with the wild-type (Fig. 4D). When comparing the effect of inoculation 16
with the wild-type strain or with the triple mutant, we observed that the KSW strain 17
appeared to biocontrol better than the wild-type strain. However, we did not observe 18
statistically significant differences (p<0.05) between KSW and the wild-type strain. 19
20
Analysis of biocontrol on strawberry. The same strains were tested for biocontrol in 21
the strawberry-Phytophtora cactorum system. This pathogenic oomycete resulted highly 22
infective in strawberry plants, especially affecting the root system but also inducing 23
symptoms in aerial parts. 15% of the plants infected were dead after 15 days and 70% of 24
the plants were infected (dead plus symptomatic plants). The most prominent symptom 25
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was root necrosis, affecting almost 60% of the surviving plants (Fig. 5B) and symptoms 1
in aerial parts were observed in 45% of surviving plants (Fig. 5A). We were able to 2
recover the pathogen from the crown in 40% of the plants (Fig. 5C) and from the root in 3
27% of the plants (Fig. 5D). Inoculation with any of the bacterial strains resulted in the 4
survival of all the plants and a clear reduction of diseased plants that was 35% for 5
inoculation with the wild-type strain, 40% for V35 and 25% for KSW. There was also a 6
reduction on the recovery of the pathogen from crown and root (Fig. 5C and D), 7
indicating that the three strains had biocontrol ability. As in the case of the tomato-8
Fusarium system, the poorer results were obtained with V35, especially on the recovery 9
of the pathogen from the crown, that was not significantly different (p<0.05) from the 10
non-inoculated plants (Fig. 5C). Inoculation with either the wild-type strain or the KSW 11
strain always resulted in results significantly better than in the uninoculated treatment. 12
When comparing the effect of inoculation of the wild-type strain and the triple mutant 13
strain KSW, this strain always showed a better performance than the wild-type and 14
differences were statistically significant (p<0.05) for the percentage of plants with 15
symptoms in aerial parts (30% for the wild-type strain versus 10% for the KSW mutant) 16
and the recovery of the pathogen from the root: P.cactorum was not recovered from any 17
plant in the case of the KSW inoculation, although it was recovered from a small 18
percentage of the plants inoculated with the wild-type strain (Fig. 5D). 19
20
DISCUSSION 21
P. fluorescens F113 is a biocontrol agent and it has been shown to inhibit in vitro the 22
growth of several fungi and oomycete, including Pythium ultimum, Phoma betae, 23
Rhizopus stolonifer and Fusarium oxysporum (35). Biocontrol activity of F113 has been 24
shown for P. ultimum in sugar beet (17) and pea (4, 27) and for Rhizoctonia solani in 25
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sugar beet (31). It has also been proposed as a biocontrol agent against the cyst 1
nematode Globodera rostoechensis in potato (13). In all these cases, biocontrol activity 2
has been directly related to the production of the fungicide DAPG. Here we have 3
extended biocontrol activity of F113 in two new pathosystems: Fusarium oxysporum 4
f.sp. radicis-lycopersici (FOL)/ Lycopersicum esculentum (tomato) and Phytophthora 5
cactorum (Pc)/ Fragaria vesca (strawberry). 6
Bacterial motility is one of the most important traits for rhizosphere colonization and is 7
likely to be involved in biocontrol. Non-motile, non-chemotactic or reduced-motility 8
mutants are among the most impaired mutants in rhizosphere competition experiments 9
(9, 36, 41). Furthermore, hypermotile phenotypic variants are selected by the 10
rhizosphere environment, occupying the most distal parts of the root (25, 32). Bacterial 11
motility can be considered a quantitative trait and mutants showing different levels of 12
swimming motility can be isolated (25). We have previously shown the polygenic 13
control of swimming motility, a trait regulated independently by at least three pathways: 14
the GacAS pathway, the SadB pathway and the WspR pathway (28). Here we show that 15
the kinB gene represses swimming motility independently of the SadB and WspR 16
pathways, since double mutants affecting kinB and either of those genes, showed an 17
additive phenotype (Fig. 1). We have also shown that kinB acts independently of the 18
gene encoding its cognate response regulator AlgB, since a mutation in the algB gene 19
had no effect in motility. It is interesting to note that in P. aeruginosa AlgB activity for 20
alginate production has been demonstrated in the absence of KinB- mediated 21
phosphorylation (23) indicating that the KinB/AlgB is an atypical two-component 22
system. Furthermore, it has been recently shown than in P. aeruginosa KinB is a major 23
determinant of virulence, regulating motility, quorum sensing and biofilm formation 24
independently of its kinase activity and of its cognate response regulator AlgB (11). On 25
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the other hand, epistasis analysis and the phenotype of double mutants showed that kinB 1
acts in the GacAS pathway, downstream of the gacAS genes. Gac mutants in 2
Pseudomonas fluorescens are impaired in secondary metabolism and are unable to 3
produce metabolites that are relevant for biocontrol such as the fungicide diacetyl-4
phloroglucinol (DAPG) (1, 42), cyanide (1, 6) and an extracellular metalloprotease (6). 5
The lack of these metabolites renders strains harboring gac mutations unsuitable for 6
biocontrol. Here we have shown that although the kinB mutation is affecting the same 7
pathway than gac mutations, kinB acts downstream the gacAS genes and a kinB mutant 8
shows a wild-type phenotype for secondary metabolism, as shown for pyoverdine and 9
exoprotease production (Fig. 2). 10
These results and the fact that a triple mutant KSW was more competitive that the wild-11
type strain for rhizosphere colonization (Fig. 3), while conserving an intact secondary 12
metabolism, prompted us to test the importance of competitive colonization for 13
biocontrol activity. It has been previously shown that efficient colonization is a pre-14
requisite for efficient biocontrol, since mutant derivatives of the phenazine-1-15
carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391, impaired in 16
competitive colonization, completely lost biocontrol ability against FOL in tomato 17
plants (12). It was therefore possible, that a hypercompetitive strain could perform 18
better than the wild-type strain for fungal biocontrol in the root. The results presented 19
here confirm this hypothesis using two pathogenicity systems: Tomato-FOL and 20
Strawberry-Pc grown under greenhouse conditions. Similar results were found for both 21
systems with V35, the strain affected in secondary metabolism as the poorer performer 22
in biocontrol for all the tested parameters. These results indicate that Gac-regulated 23
production of secondary metabolites is the major trait involved in fungal biocontrol by 24
Pseudomonas fluorescens F113. However, other Gac-independent traits should be 25
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implicated in biocontrol, since the hypermotile variant V35 which harbors a gacS 1
mutation still possesses significant biocontrol activity in both systems. Comparison of 2
the performance of the wild-type strain and the KSW mutant yielded also similar results 3
in both pathosystems: the KSW mutant showed better performance than the wild-type 4
strain. These results were especially clear in the case of Strawberry-Pc. In this system, 5
statistically significant differences between both strains were obtained for the 6
percentage of symptomatic plants and for the recovery of the pathogen from the root, 7
which was not recovered from any plant inoculated with KSW. Since both strains are 8
nearly isogenic, sharing a common genetic background, differences in performance 9
could be attributed to its differences in motility and competitive colonization. It should 10
be noted that hypermotile, hypercompetitive mutants or variants of strain F113 do not 11
differ from the wild-type strain in colonization levels, colonization sites or colonization 12
patterns when individually inoculated (5). 13
Competitive colonization performance has been used as trait for the selection of better 14
strains for biocontrol (29, 38). Here we have shown that enhancing rhizosphere 15
competitive colonization by manipulating traits known to be relevant for this process 16
may result in an improvement in biocontrol of pathogenic fungi in the root. We have 17
also shown that rational design of improved inoculants through site directed 18
mutagenesis is possible. 19
20
ACKNOWLEDGEMENTS 21
E. B. was recipient of a postgraduate research contract from Comunidad de Madrid. A. N. 22
was recipient of a FPI fellowship from Ministerio de Ciencia e Innovación (MICINN, 23
Spain). Research was funded by grant BIO2009-08254 from MICINN and the research 24
program MICROAMBIENTE-CM from Comunidad de Madrid. 25
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2. Aguirre de Cárcer, D., M. Martín, M. Mackova, T. Macek, U. Karlson, and 9
R Rivilla. 2007a. The introduction of genetically modified microorganisms 10
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1
Table 1: Strains and plasmids used.
Characteristics Reference
Strains Abbreviations
DH5α E. coli cloning strain Gibco-BRL
F113rif F113 P. fluorescens wild-type RifR (Shanahan et al., 1992)
F113gacS F113rif gacS- Rif
R Km
R (Martínez-Granero et al., 2006)
F113sadB F113rif sadB- Rif
R Km
R (Navazo et al., 2009)
F113kinB F113rif kinB- Rif
R Km
R This work
F113wspR F113rif wspR- Rif
R Spc
R (Navazo et al., 2009)
F113algB F113rif algB- Rif
R Km
R This work
F113gacS-kinB KG F113rif kinB- gacS
- Rif
R Km
R Gn
R This work
F113kinB-sadB KS F113rif kinB- sadB
- Rif
R Gn
R Km
R This work
F113gacS-sadB-wspR GSW F113rif sadB- wspR
- gacS
- Rif
R Km
R Spc
R Gn
R (Navazo et al., 2009)
F113kinB-sadB-wspR KSW F113rif sadB- wspR
- kinB
- Rif
R Km
R Spc
R Gn
R This work
F113kinB(pBG1708) F113rif kinB- pLFAR3kinB Rif
R Km
R Tc
R This work
F113v35 V35 Hypermotile phenotypic variant (Martínez-Granero et al., 2005)
Fusarium oxysporum f. IPO-DLO, Wageningen,
sp. radicis-lycopersici FOL Isolated of tomate
Netherlands
Phytophthora cactorum Pc Strawberry pathogen IFAPA, Málaga, Spain.
CH971
CH972
CH1020
CH1021
Plasmids
pGEM®T-easy vector Cloning vector AmpR Promega
pK18mobsacB Suicide vector sacB KmR (Schäfer et al., 1994)
pG18mob2 Suicide vector sacB GnR (Kirchner and Tauch, 2003)
pBG1708 pLAFR3 derivative containing the algB and kinB genes TcR This work
pRK2013 Helper plasmid KmR (Figurski and Helinski, 1979)
pRK600 Helper plasmid CmR
(Finan et al., 1986)
2
3
4
5
6
7
8
9
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1
FIGURE CAPTIONS 2
Figure 1. Swimming motility of P. fluorescens F113 and derivatives. Bacteria were 3
inoculated with a toothpick just below the surface of SA 0.3% agar plates. Swimming 4
haloes were determined after 18 h incubation at 28oC. Average swimming halo diameter 5
(mm) and standard deviations are presented. Different letters indicate statistically 6
significant differences, p<0.05. No differences in growth rates were observed for any of 7
the tested strains. The motility phenotype of the sadB and wspR mutants has been 8
previously reported (28). 9
10
Figure 2. Pyoverdine (Left) and exoprotease (Right) production by P. fluorescens F113 11
and derivatives. Pyoverdine overproduction under iron-sufficient conditions was 12
detected by fluorescence emission under U.V illumination on LB plates. Exoprotease 13
production was observed on skimmed milk plates by the appearance of a degradation 14
halo corresponding to casein degradation. 15
16
Figure 3. Competitive colonization root-tip assay: The wild-type strain was used as the 17
competitor in all the experiments. Alfalfa plants were inoculated 1:1 with the test strain 18
and the competitor and root tips were collected after 2 weeks and the bacteria present 19
resuspended and plated. Grey bars represent the percentage of colonies recovered from 20
the tested strains; black bars represent the percentage of colonies recovered from the 21
competitor (wild-type) strain. Arithmetic means and standard deviation are presented. 22
Average recovered bacteria were 3.63x106
cfu/gr root tip (fresh weight). 23
24
Figure 4. Biocontrol of P. fluorescens F113 and derivatives on the Fusarium oxysporum 25
f sp. radicis-lycopersici (FOL)/ Lycopersicum esculentum (tomato) system. A. Plant 26
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mortality; B. Plants showing symptoms in the aerial part; C. Plants showing necrotic 1
roots; D. Pathogen recovery. Different letters indicate statistically significant 2
differences, p<0.05. No dead or symptomatic plants were observed in control 3
experiments without the pathogen. 4
5
Figure 5. Biocontrol of P. fluorescens F113 and derivatives on the Phytophthora 6
cactorum (Pc)/ Fragaria vesca (strawberry) system. A. Plants showing symptoms in 7
aerial parts; B. Plants showing necrotic roots; C. Pathogen recovery from crown; D. 8
Pathogen recovery from root. Different letters indicate statistically significant 9
differences, p<0.05. No dead or symptomatic plants were observed in control 10
experiments without the pathogen. 11
12
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