quenching Rhodococcus erythropolis is revealed by a combination of

49 Pages·2011·1.27 MB·English

AEM Accepts, published online ahead of print on 11 November 2011
Appl. Environ. Microbiol. doi:10.1128/AEM.06159-11
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
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2 Efficient biostimulation of the native and introduced quorum-
3 quenching Rhodococcus erythropolis is revealed by a combination
4 of analytical chemistry, microbiology and pyrosequencing
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6 Running title: Biostimulation of quorum-quenching Rhodococcus
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8 Amélie Cirou1,2, Samuel Mondy1, Shu An3, Amélie Charrier1, Amélie Sarrazin1, Odile n
9 Thoison4, Michael DuBow3, and Denis Faure1* loa
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10 1 Centre National de la Recherche Scientifique, Institut des Sciences du Végétal, ed
11 UPR 2355, 91198 Gif-sur-Yvette, France f
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12 2 Comité Nord Plants de Pommes de Terre, CNPPT, Station de testage et m
13 d’expérimentation La Pigache, 62217 Beaurains, France h
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14 3 Université Paris-Sud, Institut de Génétique et Microbiologie, CNRS UMR 8621, p:
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15 91405 Orsay, France /a
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16 4 Centre National de la Recherche Scientifique, Institut de Chimie des Substances m
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17 Naturelles, UPR2301, 91198 Gif-sur-Yvette, France a
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19 * Corresponding author g
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20 Centre National de la Recherche Scientifique n

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21 Institut des Sciences du Végétal - UPR2355 a
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22 1, Avenue de la Terrasse h

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23 91 198 Gif-sur-Yvette, France ,

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24 Phone: [phone] 01
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25 Fax: [phone] b
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26 e-mail: [email protected] g
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27 s
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Cirou et al. Biostimulation of quorum-quenching Rhodococcus 1
28 Abstract (<250 words)
29 Degradation of quorum-sensing (QS) signals N-acylhomoserine lactones (AHL) by
30 soil bacteria is proposed as a beneficial trait for protecting crops, such as potato
31 plants, against the worldwide pathogen Pectobacterium. In this work, analytical
32 chemistry and microbial and molecular approaches have been combined to explore
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33 and compare biostimulation of the native and introduced AHL-degrading o
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34 Rhodococcus erythropolis bacterial population in the rhizosphere of potato plants lo
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35 cultivated in farm greenhouses under hydroponic conditions. We first identified e
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36 gamma-heptalactone (GHL) as a novel biostimulating agent that efficiently promotes r
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37 plant root colonization by AHL-degrading R. erythropolis population. We also h
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38 characterized an AHL-degrading, biocontrol R. erythropolis isolate R138, which was :
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39 introduced in the potato rhizosphere. Moreover, different combinations of GHL- and m
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40 R138-treatments were compared in root colonization by AHL-degrading bacteria s
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41 using a cultivation-based approach (percentage of AHL-degrading bacteria), o
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42 pyrosequencing of PCR amplified rrs loci (total bacterial community) and qPCR of the o
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43 qsdA gene that encodes an AHL-lactonase in R. erythropolis. The higher densities of
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44 AHL-degrading R. erythropolis population in rhizosphere were observed when GHL- h

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45 and R138-treatments were associated. Under this condition, the introduced R. ,

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46 erythropolis displaced the native R. erythropolis population. Finally, chemical 1
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47 analyses revealed that GHL and gamma-caprolactone (GCL), and their by-products, y

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48 gamma-hydroxyheptanoic acid and gamma-hydroxycaproic acid, rapidly disappeared e
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49 from the rhizosphere and did not accumulate in plant tissues. This integrative study
50 highlights biostimulation as a potential innovative approach for improving root
51 colonization by beneficial bacteria.
Cirou et al. Biostimulation of quorum-quenching Rhodococcus 2
52 Key words: quorum-sensing, quorum-quenching, Rhodococcus erythropolis,
53 rhizosphere, potato plant, rhizosphere, hydropony
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55
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56 Introduction w
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57 N-acylhomoserine lactones are intercellular signals used by numerous α-, β-, and γ- a
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58 proteobacteria to regulate gene expression at the population and community levels
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59 (18; 38). The mechanism connecting cell population to gene expression via AHLs is m

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60 termed quorum-sensing (QS; 17). Pectobacterium carotovorum is a causative agent tp
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61 of blackleg and soft rot diseases on several crops, including potato plants and tubers. a
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62 Over the 2004-2009 period, the quality-refusals oscillated between 2 and 5% and 3 .a
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63 and 8% of the total seed-tubers production in France and Netherlands, respectively;
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64 blackleg disease, which is caused by Pectobacterium and Dickeya, represented 10 to /

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65 30 % and 60 to 80% of the refusal motives in the same European countries. In
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66 Pectobacterium, production of virulence factors such as pectinolytic and cellulolytic rc
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67 enzymes and harpins, is positively controlled by AHLs (23; 29). In P. carotovorum, 3
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68 inhibition of AHL synthesis or degradation of the produced AHLs, results in the 0
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69 absence of the expression of the QS-regulated genes and consequently in a
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70 decrease of the virulence symptoms on potato plants (31; 23). g
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71 During the past decade, several approaches have been proposed to disrupt QS
72 regulation, and hence limit the virulence of P. carotovorum and other bacterial
73 pathogens, in which pathogenicity is controlled by QS (40; 14; 11). The anti-virulence
74 approaches targeting QS are collectively called quorum-quenching (40). Among
75 quorum-quenching (QQ) strategies targeting Pectobacterium, introduction of AHL-
Cirou et al. Biostimulation of quorum-quenching Rhodococcus 3
76 degrading bacteria remains the most explored. In the two potential QQ-agents
77 Bacillus thuringiensis and Rhodococcus erythropolis (32; 13), several AHL-degrading
78 enzymes were discovered. While B. thuringiensis expresses a unique lactonase AiiA
79 (13), R. erythropolis expresses three QQ-activities, a lactonase QsdA, an acylase
80 and a reductase (33; 28; 34). Aside from the use of biocontrol agents (32; 13), QQ-
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81 molecules (24; 27) and QQ-transgenic plants (12; 16), a biostimulation approach was o
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82 recently proposed (5; 6). It consists in the application of a biodegradable agent, lo
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83 gamma-caprolactone (GCL), to stimulate in the rhizosphere of Solanum tuberosum e
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84 the growth of endogenous AHL-degrading bacteria (hence the name biostimulation to ro
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85 describe this technique). GCL exhibits some similarity with the conserved core of the h
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86 AHL (gamma-butyrolactone) ring. Noticeably, over 95% of the GCL-stimulated QQ- :/
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87 bacteria belonged to R. erythropolis, revealing that GCL-treatment stimulated the m
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88 growth of one the most efficient QQ-bacteria. sm
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89 In this report, analytical chemistry, microbiology, and molecular approaches were g
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90 combined (i) to investigate the structural characteristics of biostimulating molecules,
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91 discover a novel biostimulation agent, gamma-heptalactone (GHL), and evaluate its r
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92 growth-stimulating effect on QQ-bacteria in the rhizosphere of potato plants, its 3
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93 impact on total bacterial community by rrs-pyrosequencing, as well as its fate by fine 2
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94 HPLC-MS/MS tools; (ii) to characterize a QQ-bacterium Rhodococcus erythropolis
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95 R138 which was used in combination or not with the biostimulating agent GHL for g
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96 enhancing the level of QQ-bacteria in the rhizosphere of potato plants in farm s
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97 greenhouse. In combined treatment GHL/R138, the fates of the introduced biological
98 and biochemical agents were analyzed, as well as the dynamics of the bacterial
99 community by rrs-pyrosequencing. This integrative approach revealed that a
100 combination of biostimulating and biological treatments is a potential, innovative
Cirou et al. Biostimulation of quorum-quenching Rhodococcus 4
101 strategy to stimulate efficiently the colonization of a crop rhizosphere by AHL-
102 degrading bacteria.
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104 Materials and Methods
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105 Chemicals and bacterial cultures w
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106 All chemicals were purchased from Sigma-Aldrich-Fluka and their structure is shown a
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107 in Figure 1A. HCA and HHA were obtained by incubating GCL and GHL,
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108 respectively, in the presence of NH4OH (0.5 M; pH 9) during 24h at 25°C for lactone m

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109 ring opening. Bacterial cultures were grown in rich media: Tryptic Soy Agar (TSA) tp
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110 purchased from AES (France) and TY (tryptone 5 g l-1, yeast extract 3 g l-1), and a
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111 synthetic medium AB (7), in which ammonium chloride (1 g l-1) was used as a sole .a
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112 nitrogen source and mannitol (2 g l-1) as a sole carbon source, except where an other
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113 carbon source is specified. P. atrosepticum CFBP 6276 was cultivated in PGA /

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114 medium (31). R. erythropolis strain R138R was cultivated in the presence of
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115 Rifampicin (Rif) at 100 mg l-1. Agar was added at 15 g l-1. rc
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116 ,

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117 Assimilation test and colorimetric quantification of GCL and GHL 9

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118 The capacity of bacterial strains to assimilate GCL and GHL in vitro was determined u
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119 by inoculating bacterial strains in medium AB supplemented with mannitol (2 g l-1) t
120 and/or GCL or GHL (2 g l-1). Bacterial growth was monitored by spectrophotometry at
121 600nm, while a colorimetric assay (39) allowed rapid quantification of the introduced
122 lactones.
Cirou et al. Biostimulation of quorum-quenching Rhodococcus 5
123
124 Identification and quantification of GCL, HCA, GHL, and HHA by HPLC-MS
125 Plant tissues were crushed under liquid nitrogen and extracted with 25 ml of
126 phosphate-buffered saline (PBS; NaCl 8 g l-1, KCl 0.20 g l-1, Na2HPO4 1.44 g l-1,
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127 KH2PO4 0.24 g l-1 and adjusted to pH 7.2) and centrifuged at 10.000 g for 20 min (4). o
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128 Plant extracts and samples from nutrient solution were filtered through n
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129 polyethersulfone columns (10kDa Vivaspin 500). Chromatographic separation of d
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130 samples (10µL) were performed by HPLC (Waters Allians 2690) conjugated with LC- fr
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131 MS/MS (Waters ZQ Mass Spectrometer with single quadrupole system and
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132 electrospray ionisation). For each of the analyzed compounds, a calibration curve is p:
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133 defined with pure compound. Mobile phase A was water/0.1% formic acid and mobile e
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134 phase B was acetonitrile/0.1% formic acid. Four experimental procedures were as
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135 defined for detecting GCL alone, GCL and its by-product HCA, GHL alone, and GHL .o
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136 and by-product HHA. o/
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137 To detect GCL molecule alone, a Waters Sunfire C18 (150mmX3mmX5µm) column a
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138 was used. The mobile phase flow rate was 0.7 ml min-1. An isocratic mixture of 70% h
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139 mobile phase A and 30% B was applied during 10 min to elute GCL and then 100% ,
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140 B during 5 min to wash the column. An equilibrate time of 10 min was allowed before 9

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141 the next injection. The retention time of GCL was 4.6 min. y
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142 GCL and its hydrolysed form HCA in a same run were detected with a Waters Gemini t
143 C18 (150mmX 2mm, 5µm) column was used. The mobile phase flow rate was 0.2
144 ml.min-1 and the retention times of GCL and HCA was 11.8 and 8.6 min, respectively.
145 A gradient was used to optimise molecule separation. The gradient started at 5% B
146 was kept at 5% for 1 min and then increased linearly to 60% in 14 min and then
Cirou et al. Biostimulation of quorum-quenching Rhodococcus 6
147 increased until to 95% in 3 min. The mobile phase composition was kept at 95% for 2
148 min and returned to 5% in 2 min. An equilibration time of 8 min was applied before
149 the next injection.
150 To detect GHL molecules alone, a Waters Gemini C18 (150mmX 2mm, 5µm) column
151 was used. Mobile phase flow rate was 0.2 ml.min-1 with a retention time of 7.3 min. D
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152 An isocratic mixture of 70% A and 30% B was applied for 10 min to elute GHL and
n
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153 then increased at 100% B during 5 min to wash the column. Before the next injection, a
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154 an equilibrate time of 10 min was allowed. d

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155 GHL and HHA in a same run were detected with the same Waters Gemini column
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156 and flow rate was used as for GHL alone. A gradient was applied to optimise p:
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157 molecule separation. The gradient started at 5% B and increased linearly to 30% in e
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158 10 min. The mobile phase composition was kept at 30% B for 10 min and increased as
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159 to 95% during 5 min and returned to 5% B in 1 min. An equilibration time of 8 min .o
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160 was allowed before the next injection. The retention time of GHL and HHA was o/
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161 respectively 17.1 and 12.6 min. M
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162
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163 Bacterial enrichment in the presence of lactones 0
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164 For comparing biostimulation of AHL-degradation by different lactones, one gram of a y

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165 non-sterile soil from the CNRS experimental field (Mérantaise) at Gif-sur-Yvette u
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166 (France) was resuspended in 10 ml of sterile 0.8% (w/v) NaCl. Soil suspensions were
167 diluted (1/50) into AB media supplemented with actidione (100 mg l-1) and mannitol –
168 as a control– or one of the tested lactones. After 2 cycles of enrichment as described
169 by Cirou et al. (5), the bacterial consortia were washed twice in 0.8% NaCl, their cell
170 density was adjusted to 1.0 at OD600, and their capacity to inactivate C6-HSL at 25
Cirou et al. Biostimulation of quorum-quenching Rhodococcus 7
171 µM and OC8-HSL at 200 nM was compared, as described by Carlier et al. (3).
172 Residual C6-HSL and OC8-HSL were quantified using biosensor strains
173 Chromobacterium violaceum CV026 (25) and Agrobacterium tumefaciens
174 NT1(pZNLR4) (4).
175 D
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176 Biocontrol tests on potato tubers n
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177 Overnight cultures of P. atrosepticum and AHL-degrading strains were washed in ed

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178 0.8% NaCl. Each tuber of S. tuberosum var. Allians was inoculated with 2.107 CFU of ro
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179 P. atrosepticum and/or 2.107 CFU of each AHL-degrading strain. Three to nine tubers h
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180 were inoculated for each condition. Five days post-infection, the tubers were cut in //
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181 the middle and photographed. Maceration symptoms were encoded as follows: 1, no m
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182 maceration; 2, moderate maceration (no more than 5mm around the infection site); 3, m
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183 strong maceration (more than 5mm). The Kruskal-Wallis test (α=0.05) allowed the r
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184 statistical analysis of the maceration categories. n

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185 rc
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186 Plant culture in hydroponic conditions in a greenhouse ,

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187 S. tuberosum var. Allians was cultivated in a greenhouse (company ‘Comité Nord’ at 9

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188 Bretteville-du-Grand-Caux, Normandy, France) under natural light at 10-15°C (night)
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189 and 25-30°C (day) from April to July. The nutritive solution Hydrobloom (Cellmax, s
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190 UK), with nitrogen at 0.8 g l-1 and potassium at 1.48 g l-1 as major components, was
191 diluted from a commercial stock solution (x250) with non-sterile water from the public
192 water system. One hundred in vitro plants were placed into holes (3 cm space to
Cirou et al. Biostimulation of quorum-quenching Rhodococcus 8
193 each other) of batch cover. Each batch (40 x 60 x 8 cm) contained 13L of nutritive
194 solution.
195
196 Analysis of cultured bacterial populations collected from potato rhizosphere
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197 One gram of roots (fresh weight) was suspended in 10 ml of sterile 10 mM MgSO4, w
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198 diluted and spread on TSA medium for counting total populations and, when lo
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199 appropriate, on AB medium supplemented with Rif at 100 mg l-1 and GCL as a sole ed

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200 carbon source for counting strain R138R only. At each time, three samples were o
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201 analyzed from each batch. From each of the samples, thirty TSA isolates were h
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202 individually cultivated on 96-microwell plates and tested for production and //
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203 inactivation of AHL on thin layer chromatography silica plate (Whatman, C18-reverse m
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204 phase), as well as growth on GCL-Rif minimal medium. AHL-production was m
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205 revealed with the biosensor A. tumefaciens NT1(pZNLR4), while C6-HSL- r
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206 disappearance (initially introduced at 25 µM) was measured with the biosensor n

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207 C. violaceum CV026, as described previously (8). a
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208 3
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209 Genotyping of cultured bacteria by rrs-sequencing 1
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210 For rrs-genotyping, the 5’-end of the rrs gene was amplified with primers pA (5’-
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211 AGAGTTTGATCCTGGCTCAG-3’) and 518r (5’-ATTACCGCGGCTGCTGG-3’), and s
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212 sequenced with pA primer by GATC (Germany). Blastn analyses were performed
213 with a 400 bp minimal length query against EMBL database
214 (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Though some sequence comparisons
Cirou et al. Biostimulation of quorum-quenching Rhodococcus 9
215 permitted identification of isolates at the species level such as R. erythropolis, the
216 genus level was retained for homogeneity.
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218 DNA extraction from potato rhizosphere
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219 The roots of two potato plants were taken from hydroponic culture batches, crushed w
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220 under liquid nitrogen and solubilised with 500µL of saline buffer (containing 10 mM lo
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221 MgSO4). Total genomic DNA of this homogenate was extracted with the appropriate ed

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222 DNA isolation Kit (MOBIO Power Soil) according to the manufacturer’s protocol. DNA o
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223 quantity and quantity was evaluated with a spectrophotometer (NanoDrop ND1000, h
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224 Labtech, France). //
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225 .a
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226 Relative abundance of qsdA gene by qPCR o
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227 In DNA extracted from rhizosphere samples, the relative abundance of the qsdA n

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228 gene of R. erythropolis was estimated by quantitative PCR (qPCR, LightCycler 480 in a
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229 96-well plates, Roche). Primers (5’-ACGAGCATGTCTTCGTTCTG and 5’- 3
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230 GGATCGACGATCGTGCTGAT) were designed for a conserved region of qsdA and 2
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231 generated a fragment of 145 bp. A calibration curve was defined with genomic DNA 9

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232 of R. erythropolis R138. Composition of the PCR mix for each sample was as follows:
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233 5µL of SYBER Green I Master Mix (Roche), reverse primer (0.5µM), forward primer s
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234 (0.5µM) and 1µL of DNA sample at 8 ng.µl-1.
235
236 Amplification of rrs target for pyrosequencing
Cirou et al. Biostimulation of quorum-quenching Rhodococcus 10