Detailed analysis of the fatty acid composition of six plant-pathogenic bacteria

Journal Pre-proofs Detailed analysis of the fatty acid composition of six plant-pathogenic bacteria Nina Wiedmaier-Czerny, Dorothee Schroth, Shiri Topman, Aya Brill, Saul Burdman, Zvi Hayouka, Walter Vetter PII: DOI: Reference:

S1570-0232(20)31330-1 https://doi.org/10.1016/j.jchromb.2020.122454 CHROMB 122454

To appear in:

Journal of Chromatography B

Received Date: Revised Date: Accepted Date:

22 July 2020 14 October 2020 2 November 2020

Please cite this article as: N. Wiedmaier-Czerny, D. Schroth, S. Topman, A. Brill, S. Burdman, Z. Hayouka, W. Vetter, Detailed analysis of the fatty acid composition of six plant-pathogenic bacteria, Journal of Chromatography B (2020), doi: https://doi.org/10.1016/j.jchromb.2020.122454

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1

Detailed analysis of the fatty acid composition of six plant-pathogenic bacteria

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Nina Wiedmaier-Czerny1, Dorothee Schroth1, Shiri Topman2, Aya Brill2, Saul Burdman3, Zvi Hayouka2* and Walter Vetter1*

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Hohenheim, D-70593 Stuttgart, Germany

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2

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Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot

Institute of Food Chemistry, Department of Food Chemistry (170b), University of

Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of

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76100, Israel

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Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot

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76100, Israel

Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of

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* Corresponding authors:

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Walter Vetter

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Phone: +49 711 459 24016

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Fax: +49 711 459 24377

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E-Mail: [email protected]

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Zvi Hayouka

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Phone: +97289489019

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Fax: +97289489483

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E-Mail: [email protected]

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Abstract

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Bacteria show distinct and characteristic fatty acid (FA) patterns which can be

30

modified by environmental conditions. In this study, we cultivated six plant-pathogenic

31

bacteria of agricultural concern and performed a detailed analysis of the fatty acid

32

composition. The study covered four strains of the gram-negative Xanthomonas

33

campestris pathovar (pv) campestris (Xcc), Xanthomonas perforans (Xp), Acidovorax

34

citrulli (AcM6) and Pseudomonas syringae pv. tomato (Pst), and two strains of the

35

gram-positive

36

Streptomyces scabies (Ssc). After cultivation by means of a standardized cultivation

37

method, freeze-dried bacteria samples were transesterified and analysed by gas

38

chromatography with mass spectrometry in full scan and selected ion monitoring (SIM)

39

modes. Altogether, 44 different FAs were detected in the six strains with individual

40

contributions of 0.01-43.8% to the total FAs. The variety in the six strains ranged

41

between 12 and 31 individual FAs. The FA composition of Xcc, Xp, Cmm and Ssc were

42

dominated by iso- and anteiso-fatty acids (especially i15:0, a15:0, i16:0), which is

43

typical for most bacteria. In contrast to this, AcM6 and Pst showed only saturated and

44

monounsaturated FAs. Four of the six bacteria showed similar FA patterns as reported

45

before in the literature. Differences were observed in the case of Cmm where many

46

more FAs were detected in the present study. In addition, to the best of our knowledge,

47

the FA pattern of Xp was presented for the first time.

Clavibacter

michiganensis

subsp.

michiganensis

(Cmm)

and

48 49

Keywords: Plant-pathogenic bacteria; fatty acid pattern; iso-/anteiso-fatty acids; X.

50

perforans; X. campestris; A. citrulli; P. syringae; C. michiganensis; S. scabies

51

2

52

1 Introduction

53

Plant-pathogenic bacteria are undesired because they can lead to food spoilage

54

[1] but also cause damage to agricultural yields [2]. According to Oerke and Dehne

55

(2004), between 18 and 32% of the worldwide crop production would be lost without

56

an active control of plant pathogens, pests and weeds [3]. Many bacteria are

57

characterized by distinct fatty acid profiles some of which can be used as diagnostic

58

marker compounds. Typical for many bacteria, especially gram-positive ones, is the

59

presence of high shares of branched-chained fatty acids (BCFAs) which are suited to

60

increase the membrane fluidity [4, 5]. Primers of BCFAs are the amino acids valine (V),

61

leucine (L), or isoleucine (I) [6, 7]. Utilization of valine leads to the even-numbered iso-

62

fatty acids (iFAs) such as i14:0 and i16:0 and utilization of leucine leads to odd-

63

numbered homologues with predominance of i15:0 and i17:0. Likewise, isoleucine is

64

the precursor of odd-numbered anteiso-fatty acids (aFAs) typically dominated by a15:0

65

and a17:0 [7]. Other bacteria take advantage of monounsaturated fatty acids

66

(monoenoic FAs) for maintaining membranes fluid, while polyunsaturated fatty acids

67

are virtually absent [7]. Fatty acid patterns of different pathogenic bacteria have been

68

reported in the past but partly with focus on only a few abundant fatty acids. Also,

69

details of the cultivation have remained unclear, and studies were difficult to compare

70

with each other.

71

Despite of their diagnostic nature, fatty acid patterns of bacteria can be affected

72

under the impact of changing environmental conditions. For instance, several bacteria

73

are known to increase the share of BCFAs and/or monounsaturated fatty acids when

74

temperatures decrease [8, 9]. These changes in the fatty acid patterns may also be

75

interpreted as a reaction to stress. Possible scenarios could range from climate change

76

to response to antibacterial compounds. Hence, the knowledge of the fatty acid pattern

3

77

of bacteria under standard conditions could serve as a starting point or reference value

78

for investigations of possible impacts on bacteria.

79

The aim of the current study was to analyse the fatty acid composition of four

80

gram-negative and two gram-positive plant pathogenic bacteria of agricultural concern

81

which were raised in culture to a certain density. The two gram-positive bacteria were

82

Clavibacter michiganensis subsp. michiganensis (Cmm) and Streptomyces scabies

83

(Ssc). Cmm is an aerobic non-sporulating plant pathogen that causes bacterial canker

84

and wilt of tomato and poses substantial economic losses to this crop [10]. Ssc is

85

another plant pathogen that causes common scab disease in potato, which results

86

either in superficial cork-like layers on the potatoes or causes the tissue to leak [11].

87

Xanthomonas campestris pathovar (pv) campestris (Xcc) is a gram-negative bacterium

88

that causes black rot disease of Brassicaceae [12]. Typical symptoms are wilting,

89

necrosis and darkening of vascular tissue [12]. Xanthomonas perforans (Xp) is one

90

among different Xanthomonas spp. that causes bacterial spot disease of tomato and

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pepper [13]. Acidovorax citrulli (AcM6) is a gram-negative, biotrophic bacterium that is

92

responsible for bacterial fruit blotch of cucurbits, especially melon and watermelon [14].

93

It causes seedling blight or fruit rot [15]. Pseudomonas syringae pv. tomato (Pst) is a

94

gram-negative, aerobic, rod-shaped and motile bacterium, which leads to bacterial

95

spot disease of tomato. The Pst strain used in this study, DC3000, is one of the best

96

studied plant pathogens [15].

97

For detailed fatty acid analysis, lipids were extracted from freeze-dried samples,

98

transesterified and the resulting fatty acid methyl esters (FAMEs) were analysed by

99

gas chromatography coupled with mass spectrometry (GC/MS) [16, 17]. The patterns

100

determined in this study were compared with literature values of the corresponding

101

bacteria, if existing.

102 4

103

2

Materials and methods

2.1

Media, solvents and chemicals

104 105 106

Nutrient broth (NB) was ordered from Difco (Detroit, US). n-Hexane (>95%),

107

methanol (99.85%), cyclohexane (99.5%) and iso-propanol (99.8%) were bought from

108

Th. Geyer (Renningen, Germany). Sulfuric acid (96%) was purchased from Carl Roth

109

(Karlsruhe, Germany). Ethyl acetate (distilled, 99.5%), pyridine (>99.9%), the silylating

110

agent

111

trimethylchlorosilane (TMCS), 99:1 (v/v) and the hydroxy-fatty acid (OH-FA) standard

112

3-hydroxyhexadecanoic acid (3-OH-16:0 (98%)) were ordered from Sigma Aldrich

113

(Steinheim, Germany). Undecanoic acid (>97%) was purchased from Fluka

114

(Steinheim, Germany). Standards of iFAs and aFAs for identification were bought from

115

Larodan (Malmö, Schweden) [18] while all other fatty acids were determined by means

116

of the Supelco 37 component FAME mix (Sigma Aldrich, Steinheim, Germany) [18].

consisting

of

N,O-bis(trimethylsilyl)trifluoroacetamide

(BSTFA)

and

117 118

2.2

Cultivation and treatment of bacterial samples

119

Strains of Xcc, Xp, Ssc, Cmm, AcM6, and Pst are described in Table S1.

120

Bacterial cultivation was performed by standard protocols [19]. In brief, bacteria were

121

grown in four 50 mL batches using nutrient broth medium (section 2.1) (28 °C, with

122

180 rpm shaking) over night. Then, batches of the same bacteria were united, diluted

123

to an OD (600 nm) of 0.1 in fresh nutrient broth medium (~108 CFU/mL) and divided

124

into different aliquots of 0.5 L each. After 24 h in a shaker (28 °C, 180 rpm), bacteria

125

were centrifuged (15 min, 8000 rpm), washed 3 times with phosphate buffered saline

126

and followed by two additional washing steps with sterile double-distilled water. Then,

127

bacteria were re-suspended in double-distilled water and freeze-dried before

128

proceeding with lipid analysis. 5

129 130

2.3 Generation of fatty acid methyl esters (FAMEs) from bacterial lipids

131

An aliquot of freeze-dried bacteria (about 10 mg per sample) was used for

132

conversion of the bacterial fatty acids in the corresponding methyl esters (FAMEs) by

133

means of 2 mL 1% sulfuric acid in methanol [17]. Each sample was heated for 90 min

134

to 80 °C, followed by 10 min ultrasonification and heated again for 30 min to 80 °C.

135

After cooling on ice, 1 mL demineralized water, 1 mL aqueous saturated NaCl solution

136

and 2 mL n-hexane were added to the reaction tube. After shaking and phase

137

separation, the upper phase was removed and transferred into a 1.5-mL amber glass

138

vial. The samples were diluted to a final concentration of ~70 µg FAMEs per mL n-

139

hexane for measurement. Additionally, an internal standard (ISTD), 11:0 ethyl ester

140

(11:0-EE), was added to the measuring solution (c = 1 µg/mL). This ISTD was

141

previously transesterified in the same way as for the bacterial samples except that

142

2 mL ethanol with 1% sulphuric acid was used instead of 2 mL methanol with 1%

143

sulphuric acid [17].

144

Alternatively, lipids were extracted from freeze dried bacteria (~100 mg) by

145

accelerated solvent extraction (ASE) using a Dionex ASE 350 (Thermo Scientific,

146

Waltham, Massachusetts, USA) instrument using the instrumental parameters of

147

Weichbrodt et al. (i.e. temperature: 125 °C, pressure: 10 MPa, heat: 6 min, static: two

148

cycles of 10 min each, flush: 60% and purge: 1 MPa N2 for 2 min) [20]. Three different

149

solvent systems (solvent system 1: 40 mL n -hexane/iso-propanol (3:2, v/v); solvent

150

system 2: 40 mL of the azeotropic mixture of cyclohexane/ethyl acetate (46:54, w/w);

151

solvent system 3: 40 mL of the azeotropic mixture of methanol/ethyl acetate (44:56,

152

w/w)) modified from Hauff and Vetter [21]. The three extracts were combined and the

153

solvent was removed using a rotary evaporator. The residue was taken up in a small

154

volume of n-hexane and transferred into a 4 mL vial. Finally, the volume was adjusted 6

155

with n-hexane to 4 mL. An aliquot of the resulting ASE lipid extract, containing between

156

100 and 300 µg fat, was transesterified as described above.

157 158

2.4 Gas chromatography with mass spectrometry (GC/MS)

159

FAMEs were analysed on a 5890 Series II Plus/5972 GC/MS system (system I)

160

using helium (purity 5.0) as the carrier gas at 1 mL/min [22]. A 60 m x 0.25 mm i.d.

161

capillary column coated with 0.1-µm film thickness 90% biscyanopropyl, 10%

162

cyanopropylphenyl polysiloxane (Rtx-2330, Restek, Bad Homburg, Germany) was

163

used according to Eibler et al. [22]. A 7673 autosampler (Hewlett-Packard/Agilent,

164

Waldbronn, Germany) was used for the injection of standard and sample solutions

165

(1 µL) into a split/splitless injector operated in splitless mode and heated to 250 °C.

166

Samples were measured in both full scan mode (m/z 50-550) and in selected ion

167

monitoring (SIM) mode using m/z 74, 79, 81, 87, 88 and 101 according to Thurnhofer

168

et al. (2008) [16].

169

Trimethylsilylated 2- and 3-hydroxy-fatty acid methyl esters (2- and 3-TMS-O-

170

FAMEs) were analysed on a 6890/5973 N GC/MS system (system II) using helium

171

(purity 5.0) as the carrier gas at 1 mL/min [23]. A pre-column (2 m × 0.25 mm i.d.,

172

deactivated with 1,3-diphenyl-1,1,3,3-tetramethyldisilazane; BGB Analytics, Böckten,

173

Switzerland) was used in combination with a 30 m x 0.25 mm i.d. HP-5MS column and

174

the temperature program of Hammann and Vetter (2016) [23]. Injections (1 µL) were

175

made with an MPS 2 autosampler (Gerstel, Mühlheim, Germany) in splitless mode and

176

heated to 250 °C. Sample and standard solutions were measured in full scan mode

177

(m/z 50-650).

178 179

2.5 Evaluation of the GC/MS measurements and reporting

7

180

Fatty acids were determined as methyl esters (FAMEs) using the 37 component

181

FAME mix and an iso-/anteiso-FAME mix as reference standards [18]. In GC/MS-SIM

182

mode, saturated FAMEs were determined by means of m/z 87 and monounsaturated

183

FAMEs with m/z 74 [24] (Table S2). Methyl esters of 3-hydroxy-FAs (3-OH-FAMEs)

184

were identified in full scan mode by initial extraction of the diagnostic fragment ion m/z

185

103 [25] from the total ion current using GC/MS system I and methyl esters of 2-

186

hydroxy-FAs (2-OH-FAMEs) by the fragment ion [M-59]+. Determination in GC/MS-

187

SIM mode of OH-FAMEs was based on m/z 74 as the quantification ion. Due to the

188

low response of 2-OH-FAMEs to m/z 74 (see below), the relevance of 2-OH-12:0-ME

189

was determined by correction with the factor of the abundance of m/z 90 to m/z 74 of

190

4.67 as determined in full scan mode. In addition, OH-FAMEs were separated from the

191

non-OH-FAMEs by means of adsorption chromatography on 0.8 g activated silica in a

192

Pasteur pipette using the method of Jenske and Vetter (2009) [26]. After separation,

193

aliquots of the OH-FAME fraction (fraction 2, gained with 6 mL ethyl acetate [27]) were

194

silylated with 50 µL BSTFA/TMCS (99:1, v/v) and 25 µL distilled pyridine [28, 29] and

195

measured on GC/MS system II. 3-TMS-O-FAMEs were studied by means of the

196

diagnostic fragment ion at m/z 175 along with [M-15]+, because the molecular ion (M+)

197

is not detectable [30]. Semi-quantitative amounts of OH-FAMEs were determined after

198

trimethylsilylation by means of the response factor of a quantitative solution of 3-TMS-

199

O-16:0-ME standard solution.

200

Positions of double bonds of monounsaturated FAs were specified if the isomer

201

was present in the reference standard. If no reference standard was available, the

202

exact position of a double bond could not be determined. Then, a list of all isomers in

203

the different samples was compiled and numbers were assigned with increasing

204

retention time (for example 18:1 (#1)), which was applied to all bacteria samples. From

205

GC elution rules it is known that isomers elute the earlier the closer the double bond is 8

206

to the carboxylic chain. Unknown FAs were assigned to saturated or monounsaturated

207

FAs depending on the ratio of m/z 74 to 87 and then were evaluated with m/z

208

mentioned above [24].

209

All fatty acids were determined as FAMEs, and OH-FAs additionally after

210

silylation (TMS-O-FAMEs). For reason of simplicity, they will be later mostly reported

211

and discussed as “fatty acids”. Also, identified fatty acids will be listed by short term

212

while isomers without full verification were labelled with numbers according to the GC

213

elution order.

214 215

2.6 Quality control

216

All data of the second cultivation were based on two independent sample

217

preparations and measurements of aliquots of the freeze-dried materials (n = 2). Low

218

standard deviations between duplicates (< 1% except one case of 1.09%) verified the

219

good precision of the method. Hence, mean values of the % contribution to the total

220

fatty acids will be reported in the following. The good precision of the present GC/MS-

221

SIM method was also shown in a previous paper [24]. Linearity of calibration lines for

222

several FAMEs exemplarily tested resulted in a coefficient of determination of > 99%.

223

Due to small amounts of the bacteria samples, the samples of the first cultivation (Xcc

224

1 and AcM6 1) could only be analysed once (single determination). Therefore, larger

225

amounts of bacteria were cultivated in the second part of this study and these samples

226

were considered as “main sample” and will thus be discussed first. Yet, the impact of

227

cultivation on the FA profiles was tested by comparing the results of the first and

228

second cultivation of Xcc and AcM6 which took place after a gap of six months (section

229

3.2.1 and 3.2.2).

230 231

3 Results and discussion 9

232 233

3.1 Analytical characteristics of the GC/MS method

234 235

Structures of conventional fatty acids were confirmed by authentic reference

236

standards except some isomers of monoenoic fatty acids which were assigned by

237

means of GC retention time and the characteristic fragmentation pattern according to

238

Thurnhofer and Vetter [24] (section 2.5). The GC/MS-SIM method of Thurnhofer and

239

Vetter for conventional FAMEs is based on low-mass fragment ions, while the low

240

abundant molecular ions were not recorded [24]. Next to retention times, unequivocal

241

assignment of FAMEs to groups of saturated, monoenoic and polyunsaturated (not

242

detected in this study) fatty acids was based on abundance ratios of the SIM ions [31,

243

32]. Within the group of saturated FAMEs, co-elutions could be excluded because of

244

the known elution order and distinct peak pattern of iso < anteiso < straight chained

245

isomer while otherwise branched fatty acids were not present because all retention

246

times could be traced back to saturated FAMEs present in the standards. Co-eluting

247

pairs of monounsaturated FAMEs could not be excluded and a full structural

248

assignment was not possible due to the lack of reference standards. Therefore,

249

isomers of unsaturated FAMEs were labelled with numbers according to (increasing)

250

GC retention times (section 2.5). Co-elutions between saturated and monoenoic fatty

251

acids were not observed in this study.

252

Limits of detection (LOD) based on signal to noise ratio (S/N) > 3 and limits of

253

quantitation (LOQ) based on S/N > 10 were determined for all FAMEs present in both

254

the samples and the 37K FAME standard and selected iso- and anteiso-FAMEs. LOQ

255

values of saturated FAMEs ranged from 8-19 pg, respectively (Table 1). These values

256

were in the same range as those exemplarily reported by Thurnhofer and Vetter,

257

namely 20 pg for the fatty acid with the lowest response [24]. LOQ of monoenoic fatty 10

258

acids was in the range of 26-54 pg (Table 1). These values were higher since the

259

contribution of m/z 74 to the total ion current of GC/MS chromatograms of

260

monounsaturated FAMEs was lower (due to their stronger fragmentation) compared to

261

m/z 87 of saturated FAMEs [24, 31, 32].

262

In the literature, OH-fatty acids were frequently quantified as FAMEs after

263

additional silylation [33, 34] or after formation of other derivatives [27, 34]. Using the

264

present chromatographic conditions, peak shapes of free OH-fatty acids were suited

265

for direct determination while silylation was used for initial verification only (see below).

266

Stability of OH-FAMEs in the solutions was tested by measuring the same sample on

267

different days. Quantitation resulted in almost identical results for conventional and

268

OH-FAMEs. Therefore, the method precision could be described by means of the

269

relative standard deviation which ranged between 1 and 6% depending on the

270

concentration of fatty acids in the sample solutions. In contrast to OH-FAMEs, silyl

271

ethers of OH-FAMEs are stable for only a few days.

272

Free 3-OH-FAMEs were identified by means of the diagnostic base peak at

273

m/z 103 [25] (Fig. 1a), which is formed by cleavage between C-3 and C-4 (which

274

corresponds with m/z 87 in the GC/MS spectra of conventional FAMEs). This

275

fragmentation is particularly favoured in the GC/MS spectra of 3-OH-FAMEs because

276

it represents the -ion relative to the hydroxyl group on C-3 (Fig. 1e). Further

277

verification of OH-FAMEs was obtained after adsorption chromatographic separation

278

of conventional FAMEs on activated silica [26] (section 2.5). Since molecular ions of

279

3-OH-FAMEs could not be detected (Fig. 1a), structural assignments of homologues

280

in GC/MS-SIM chromatograms were additionally based on the following procedure.

281

Injection of a standard of 3-OH-16:0-ME and 16:0-ME indicated a difference in

282

retention time (tR) of 8.29 min. Such a difference in tR was used to tentatively assign

283

structures to 3-OH-FAMEs in the samples. Plots of logarithmic retention times over 11

284

carbon chain lengths were used to assign families of OH-FAs, similarly to previous

285

approaches with branched-chain fatty acids [35, 36]. Moreover, m/z 175 in the silylated

286

GC/MS spectrum of 3-TMS-O-12:0-ME corresponded with m/z 103 in the free form

287

(Fig. 1a, c). By this measure, up to 17 3-OH-FAs could be identified in the bacterial

288

samples.

289

Interestingly, sample Pst featured one FAME that eluted into the silica fraction

290

of OH-FAMEs (section 2.5) but did not form the diagnostic base peak at m/z 103 (Fig.

291

1b). The weak molecular ion at m/z 230 indicated the presence of an isomer of 3-OH-

292

12:0-ME. This was further substantiated after silylation of the sample (Fig. 1c, d) by

293

means of m/z 287 which corresponds with the [M-15]+ fragment ion of TMS-O-12:0-

294

ME. Formation of the McLafferty ion at m/z 90 (Fig. 1b, h) instead of m/z 74 of both

295

conventional FAMEs and 3-OH-FAMEs (Fig. 1a, g) produced strong evidence that the

296

OH group was located on C-2. The structure of 2-OH-12:0-ME was further

297

substantiated by m/z 171 [M-59]+ which is formed by -cleavage based on the

298

molecular ion formed in the OH-moiety (Fig. 1f). The corresponding [M-59]+ fragment

299

ion was also detected in the GC/MS spectrum of (silylated) 2-TMS-O-12:0-ME at m/z

300

243 (Fig. 1d). Last not least, Keinänen et al. showed that 3-OH-FAs eluted slightly

301

earlier than 2-OH-FAs from DB5-like columns [25]. This is in agreement with our

302

results. Noteworthy, 2-OH-12:0 was the only 2-OH-fatty acid detected in any of the

303

bacterial samples (see below). Its abundance was determined by means of m/z 74

304

followed by correction by means of the factor of the peak area of m/z 90 to m/z 74 of

305

4.67 (section 2.5).

306

The limit of detection of OH-FAMEs was exemplarily studied by means of 3-OH-

307

16:0-ME which was available as reference standard (section 2.1). In its free form, using

308

m/z 74 as quantification ion, the LOQ was found to be 80 pg and the LOD 24 pg (Table

309

1). Accordingly, the LOD was in the range of values of 7-50 pg reported for 3-OH-FAs 12

310

in the literature [37]. While LOD/LOQ of our method could be lowered by selecting the

311

more abundant m/z 103 as quantification ion (Fig. 1a), selecting m/z 74 was finally

312

favoured because this did not necessitate the addition of a further ion (here: m/z 103)

313

to the GC/MS-SIM method. Based on these data and the sample preparation scheme,

314

LOQ for FAMEs in sample solutions corresponded with 6-66 ng/mg dry weight of

315

bacterial samples (Table 1).

316 317

3.2 Fatty acid patterns of the six bacteria cultured in this study

318

Fatty acids were determined in four gram-negative bacteria (Xanthomonas

319

campestris pathovar (pv) campestris (Xcc), Acidovorax citrulli (AcM6), Xanthomonas

320

perforans (Xp), Pseudomonas syringae pv. tomato (Pst), section 3.2.1-4) and two

321

gram-positive bacteria (Clavibacter michiganensis subsp. michiganensis (Cmm),

322

Streptomyces scabies (Ssc), section 3.2.5-6). Altogether, 44 different FAs were

323

detected in the six bacterial strains with individual contributions of 0.01–43.8% to the

324

total FAs. The variety in the six bacteria ranged between 12 and 31 individual FAs.

325 326

3.2.1 Fatty acid pattern of Xanthomonas campestris pathovar (pv) campestris

327

(Xcc).

328

Cultivation of Xcc was carried out twice (Xcc 1 and Xcc 2) using the same starter

329

and conditions. The sample of the second treatment (Xcc 2) featured twenty-nine fatty

330

acids which contributed between 0.1% and 23.7% to the total FAs (Table 2; Fig. S1).

331

Highest shares originated from i15:0 (23.7%) followed by a15:0 (16.6%), 16:1n-7 (#2)

332

(11.1%) and 16:0 (10.2%). Further abundant FAs were i17:0 (6.3%), 15:0 (6.1%), a17:1

333

(#2) (4.9%), i11:0 (4.2%), 17:1n-8 (#5) (3.3%) and i16:0 (3.1%). 16:1n-9 (#1) and 14:0

334

contributed to 2.0% and 1.8% to the fatty acid pattern (Table 2; Fig. S1). Shares of

335

0.4-1% were determined for 17:0, i14:0, a17:0, 18:1n-9 (#4), 10:0, a15:1 (#1), 18:1n-7 13

336

(#5), and 3-OH-FA (#2) (Table 2; Fig. S1). According to the approach presented in

337

section 2.5, this FA was tentatively identified as 3-OH-i11:0. Finally, traces only (i.e.

338

0.1-0.4%) were found for a11:0, 12:0, i13:0, a13:0, 13:0, 18:0, 17:1 (#6), 3-OH-12:0

339

(#3), and 3-OH-i13:0 (#4) (Table 2; Fig. S1). Due to the lack of reference standards,

340

families of 3-OH-FAs were assigned by plots of log tR over the carbon chain length

341

[35]. Because of the wide elution range and the impact of the GC oven program, graphs

342

were split into two groups, i.e. one displaying homologs with ten to 13 carbons and the

343

other those with 14 to 17 carbons (Fig. 2a, b). This procedure resulted in three straight

344

lines for unbranched saturated 3-OH-FAs, 3-OH-iFAs and 3-OH-aFAs (Fig. 2).

345

The second sample of Xcc (Xcc 1) was cultivated and analysed half a year

346

before Xcc 2 using the same growing conditions. While the variety of FAs was almost

347

the same, there were some remarkable differences in the abundance ratios (Table 2).

348

For example, i15:0 amounted to 34.5% in Xcc 1 compared to 23.7% in sample Xcc 2

349

as discussed above. This was compensated by lower amounts of i11:0 (0.4% in Xcc 1

350

and 4.2% in Xcc 2), 16:0 (7.1% vs. 10.2%), i17:0 (3.9% vs. 6.3%) and 17:1n-8 (#5)

351

(1.7% vs. 3.3%) in Xcc 1 (Table 2). Likewise, the abundance ratio of minor fatty acids

352

was different. Unfortunately, the reasons for the variations in the FA patterns of the two

353

treatments could not be determined. However, the fatty acid patterns of both

354

treatments were in the range of those determined in 20 species of Xanthomonas

355

including Xcc in the literature [38] (Table 2). Compared to the present study, Vauterin

356

et al. detected small amounts (<1%) of additional 3-OH-FAs (3-OH-10:0/ -11:0/ -13:0/

357

-i12:0/ -i17:0) (Table 2). Silylation of the FAME fraction converted 3-OH-FAMEs into

358

the corresponding 3-TMS-O-FAMEs enabled us to verify the presence of these and

359

additional 3-OH-FAs in the samples but at even lower levels (Table S3).

360 361

3.2.2 Fatty acid pattern of Acidovorax citrulli (AcM6). 14

362

AcM6 was also cultivated twice. The second sample (AcM6 2) showed sixteen

363

FAs, which contributed with 0.01-42.7% to the total FAs (Table 3; Fig. S2). AcM6

364

featured almost exclusively saturated and monounsaturated FAs in similar shares

365

(Table 3; Fig. S2). Moreover, the two main FAs, i.e. 16:0 (42.7%) and 16:1n-7 (#2)

366

(39.3%), contributed with >80% to the total FAs of AcM6 (Table 3; Fig. S2). In addition,

367

18:1n-7 (#5) (7.6%), 14:0 (3.2%) and 12:0 (2.9%) were present at medium abundance

368

while 10:0, 15:0, 17:0, 18:0, 16:1n-9 (#1), 18:1 (#3), 18:1n-9 (#4), 3-OH-10:0 (#1)

369

contributed less than 2% and i15:0, a15:0 and i16:0 only at 0.01-0.02% to the total FAs

370

(Table 3).

371

The FA pattern of the first cultivation (AcM6 1) showed only small differences

372

although the sample preparation methods were different (direct transesterification in

373

sample 2 vs. ASE extraction in sample 1, Table 3). Moreover, Walcott et al. (2000) [39]

374

analysed 14 haplotypes of AcM6 and reported a very similar FA pattern as in the

375

present work (Table 3). Again, the variety and abundance of 3-OH-FAs (3-OH-10:0/ -

376

11:0/ -12:1/ -12:0 at 0.1-8% [39]) was higher than in the present study (3-OH-10:0 at

377

1.0% and traces of two further 3-OH-FAs after silylation, Table 3, Table S3).

378 379

3.2.3 Fatty acid pattern of Pseudomonas syringae pv. tomato (Pst).

380

The FA pattern closely resembled the one of AcM6. However, Pst contained

381

even less, i.e. only 14 different FAs which contributed between 0.02% and 41.9% to

382

the FA pattern (Table 4; Fig. 3a). Also in the case of Pst, 16:0 (33.2%) and 16:1n-7

383

(#2) (41.9%) presented more than 3/4th of the total FAs. In addition, 18:1n-7 (#5)

384

(14.4%), 12:0 (5.02%) – as in AcM6 – along with 18:0 (1.95%), 3-OH-10:0 (#1) (1.05%)

385

and 18:1 (#2) (0.93%) contributed with around 1% or more to the total fatty acids (Table

386

4; Fig. 3a). Less than 1% of the total fatty acids originated from 10:0, 14:0, 17:0, 18:1n-

15

387

9 (#4) and 2-OH-12:0 as shown in section 3.1 (Table 4; Fig. 3a), respectively, along

388

with traces of iFAs and aFAs, i.e. 0.03% a13:0 and 0.02% i16:0 (Table 4).

389

The FA pattern was similar to those reported in different Pseudomonas strains

390

including one of Pst [40] (Table 4). For instance, 16:0 (26.0%) and 16:1n-7 (#2)

391

(40.5%) were also the predominant two fatty acids [40]. Stead detected higher amounts

392

of OH-FAs including 2-OH-12:0 (between 2.6–4.0%) of three OH-FAs than in the

393

present sample (Table 4) [40]. Semi-quantitative levels determined after silylation were

394

in the range 1-190 µg/g dry weight, respectively (Table S3). Noteworthy, 2-OH-12:0 in

395

Pst was the only -hydroxy fatty acid detected in any of the samples (section 3.1).

396 397

3.2.4 Fatty acid pattern of Xanthomonas perforans (Xp).

398

Xp showed a more complex FA pattern with 29 different FAs (0.1–19.1%

399

contribution to the FA pattern) (Table 5; Fig. 3b). The main FAs were i15:0 (19.1%) >

400

16:0 (14.8%) > 16:1n-7 (#2) (13.8%) > a15:0 (11.2%) > i17:0 (10.3%) (Table 5; Fig.

401

3b). In addition, i11:0, i13:0, a13:0, i14:0, 14:0, 15:0, i16:0, a17:0, 17:0, 16:1n-9 (#1),

402

i17:1 (#1) and 17:1n-8 (#5) contributed with 1-4%, respectively, to the total fatty acids

403

(Table 5; Fig. 3b). Small amounts (<1%) of 10:0, a11:0, i12:0, 12:0, 13:0, 18:0, 17:1

404

(#6), 18:1n-9 (#4), 18:1n-7 (#5), three 3-OH-FAs (#2-4) (3-OH-i11:0/ -12:0/ -i13:0) and

405

traces of further 3-OH-FAs (detected after silylation) were also present in the sample

406

(Table 5; Table S3). Unfortunately, previous reports on the FA pattern of X. perforans

407

could not be identified in the scientific literature.

408 409

3.2.5 Fatty acid pattern of Clavibacter michiganensis subsp. michiganensis

410

(Cmm).

411

The most complex FA pattern was observed in this bacterium. Namely, thirty-

412

one FAs contributed with 0.02-37.8% to the total FAs (Table 6; Fig. 3c). This was 16

413

surprising, because Gitaitis and Beaver (1990) [41] only listed seven fatty acids in other

414

strains of this bacterium, i.e. a15:0 (40.9%), a17:0 (21.3%), i16:0 (13.9%) along with

415

12:0, a15:1, i15:0, i16:0 and 16:0 (Table 6) [41].

416

The FA pattern of the present sample of Cmm was dominated by i15:0 (37.8%)

417

followed by 16:0 (16.0%) and a15:0 (11.3%) (Table 6; Fig. 3c). Notable, but smaller

418

contributions of 5.4% originated from an i11:0 (assignment based on tR according to

419

Schröder and Vetter [36]). In addition, i17:0 (5.0%), 14:0 (4.0%), 17:1 (#4) (3.8%),

420

16:1n-9 (#1) (2.8%) and a17:1 (#2) [35] (2.2%) were detected along with five fatty acids

421

at 1-2% abundance, i.e. 15:0, i16:0, a15:1 (#1) [42], 16:1n-7 (#2), 17:1 (#7) (Table 6;

422

Fig. 3c). Less than <1% was determined for 10:0, i13:0, i14:0, a17:0, 18:1n-9 (#4) and

423

3-OH-i11:0 (#2) (Table 6; Fig. 3c) while traces (0.02-0.3%) originated from a11:0, 13:0,

424

a13:0, 17:0, 18:0, i19:0, 18:1n-7 (#5), 3-OH-i13:0 (#4) and one unknown saturated FA

425

and two unknown monounsaturated FAs (assignment based on the occurrence and

426

ratios of m/z 74 and 87 [24]). After silylation six more 3-OH-FAs (i.e. seven in total)

427

were detected, but only in small amounts, i.e. between 4 and 45 µg/g dry weight (Table

428

S3; Fig. 4).

429

The deviations between the present FA profile in Cmm and the one reported by

430

Gitaitis and Beaver (1990) [41] (Table 6) could be due to (i) different cultivation

431

conditions, (ii) different strains and/or (iii) the more thorough analysis with additional

432

focus on minor fatty acids in this study.

433 434

3.2.6 Fatty acid pattern of Streptomyces scabies (Ssc).

435

GC/MS analysis indicated the presence of 18 fatty acids, which contributed with

436

0.1-26.1% to the fatty acid pattern (Table 7; Fig. 3d). Three FAs represented about

437

2/3rd of the fatty acids, i.e. a15:0 (26.1%), i16:0 (22.2%) and a17:0 (16.0%) (Table 7;

438

Fig. 3d). In addition, i15:0 (8.1%), 16:0 (7.9%), i14:0 (3.4%), 17:1 (#4) (3.4%) and 18:1 17

439

(#1) (1.8%) were present in higher amounts (Table 7; Fig. 3d). Accordingly, the

440

present bacterium featured only FAs with 14 or more carbon atoms. In agreement with

441

that, Ndowora et al. (1996) [43] and Paradis et al. (1994) [37] determined similar fatty

442

acid patterns in literature samples of pathogenic Ssc except some shifts in the

443

abundance order (Table 7) [43, 44].

444 445

4 Conclusion

446

The FA patterns of four of the six bacteria were very similar to the those reported

447

in the literature. Differences were observed in the case of Cmm where a higher number

448

of fatty acids was detected in our study. In addition, to the best of our knowledge, the

449

FA pattern of Xp had not been reported before in the literature. Our repeated cultivation

450

of two bacteria after several months showed some noticeable differences in the pattern

451

of Xcc. Such variations should be taken into account when different studies or

452

cultivations are compared with each other or in studies on the impact of external factors

453

on fatty acid patterns of bacteria.

454 455

Conflict of interest

456

The authors declare that they have no conflict of interest.

457 458

Acknowledgement

459

We are grateful for financial support by the Ministry of Science, Research and Arts,

460

Baden-Württemberg (Germany) in the framework of the partnership program between

461

the Robert H. Smith Faculty of Agriculture, Food and Environment of the Hebrew

462

University of Jerusalem and the Faculties of Agricultural and Natural Sciences of the

463

University of Hohenheim.

464 18

465

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Phytopathology 86 (1996) 138-143.

661 662

[44] E. Paradis, C. Goyer, N.C. Hodge, R. Hogue, R.E. Stall, C. Beaulieu, Fatty acid

663

and protein profiles of Streptomyces scabies strains isolated in Eastern Canada,

26

664

International Journal of Systematic and Evolutionary Bacteriology 44.3 (1994) 561-

665

564. https://doi.org/10.1099/00207713-44-3-561

666 667

27

668

Captions to Figures

669

Fig. 1. GC/MS mass spectrum of (a) 3-OH-12:0-ME, (b) 2-OH-12:0-ME, (c) 3-TMS-O-

670

12:0 and (d) 2-TMS-O-12:0 with specific ions. Additional displays show the structures

671

of 3-OH-12:0-ME (e) and 2-OH-12:0-ME (f) and the mechanism of the formation of the

672

McLafferty ions (g, h).

673 674

Fig. 2. Logarithmic retention times (log tR) plotted over the carbon number of families

675

of saturated TMS-O-FAMEs (i.e. unbranched FAs, iFAs and aFAs) with (a) range C10-

676

C13 and (b) C14-C17.

677 678

Fig. 3. GC/MS full scan chromatograms of fatty acid methyl esters of (a) Pseudomonas

679

syringae pv. tomato (Pst), (b) Xanthomonas perforans (Xp), (c) Clavibacter

680

michiganensis subsp. michiganensis (Cmm) and (d) Streptomyces scabies (Ssc) and

681

of the internal standard 11:0-ethyl ester (11:0-EE).

682 683

Fig. 4. (a) GC/MS ion chromatogram (m/z 175) of the hydroxy-fatty acid fraction of

684

Clavibacter michiganensis subsp. michiganensis (Cmm) obtained after solid phase

685

fractionation followed by silylation into the corresponding TMS-O-FAMEs with (b) a

686

structure of 3-TMS-O-i13:0 and (c) the mass spectrum of 3-TMS-O-i13:0 with

687

characteristic ions.

688

28

29

689

690

30

691

31

692 693

32

694 695

Table 1: Limit of quantitation (LOQ) and limit of detection (LOD) for different FAMEs in pg.

696

FAME LOQ S/N 10 (6) [pg] LOD S/N 3 [pg] 10:0 8.9 2.7 11:0 13 3.9 12:0 11 3.2 13:0 8.4 2.5 14:0 9.2 2.8 14:1 27 8.1 15:0 10 3.1 15:1n-5 26 7.7 16:0 18 5.4 16:1n-7 39 12 17:0 17 5.2 17:1n-7 28 8.5 18:0 9.5 2.8 18:1n-9 54 16 i18:0 19 5.6 a18:0 19 5.6 3-OH-16:0 80 (48) 24 * Theoretical concentration of the FAs in Cmm at S/N 10 (6)

697

33

c [ng/mg] * 6 8 6.4 7.4 8.7 19 16 32 16 25 11 47 12 13 66 (39)

698 699 700 701

Table 2: Average of percentage composition of the FAs of Xanthomonas campestris pv. campestris (Xcc, n = 2). Comparison of the present sample (Xcc 2), the sample cultivated half a year ago (Xcc 1) and of Xcc sample analysed by Vauterin et al. (1996) [38]. FAME variety 10:0 i11:0 a11:0 i12:0 12:0 i13:0 a13:0 13:0 i14:0 14:0 i15:0 a15:0 15:0 i16:0 16:0 i17:0 a17:0 17:0 18:0 14:1 (#1) 14:1 (#2) i15:1 a15:1 (#1) 15:1 16:1n-9 (#1) 16:1n-7 (#2) a17:1 (#2) 17:1n-8 (#5) 17:1 (#6) 18:1n-9 (#4) 18:1n-7 (#5) 3-OH-10:0 (#1) 3-OH-i11:0 (#2) 3-OH-11:0 3-OH-i12:0 3-OH-12:0 (#3) 3-OH-i13:0 (#4) 3-OH-13:0 3-OH-i17:0 Instrument Column

Xcc 1 [%] 32 0.02 0.4 0.04 0.02 0.03 1.0 0.2 0.1 1.1 2.1 34.5 19.5 6.0 2.2 7.1 3.9 0.5 0.4 0.6 0.04 0.02

Xcc 2 [%] 29 0.7 4.2 0.4

0.7 0.1 1.2 11.2 4.7 1.7 0.1

0.6

0.1 0.3 0.1 0.1 0.8 1.8 23.7 16.6 6.1 3.1 10.2 6.3 0.8 0.9 0.1

Xcc by Vauterin et al. (1966) [38] [%] 26 0.6 (± 0.3) 4.5 (± 0.7)

0 (± 0.3) 0.7 (± 0.5) 0.8 (± 0.4) 26.5 (± 3.4) 13.9 (± 2.2) 1.2 (± 0.6) 3.2 (± 1.3) 3.6 (± 1.0) 6.8 (± 1.4) 0.8 (± 0.5)

0.4 (± 0.4)

0.04

2.0 11.1 4.9 3.3 0.2 0.7 0.5

0.2

0.5

0.04 0.2

0.1 0.1

GC/MS 60 m x 0.25 mm i.d. 90% biscyanopropyl, 10% cyanopropylphenyl polysiloxane capillary column

34

0.6 (± 0.4) 0.9 (± 0.7) 12.7 (± 2.0) 1.4 (± 0.5) 0.2 (± 0.3) 0.0 (± 0.1) 2.8 (± 0.4) 0.1 (± 0.2) 0.2 (± 0.3) 2.6 (± 0.5) 4.7 (± 0.7) 0.3 (± 0.3) 0.1 (± 0.2) Gas-liquid chromatography 25 m x 0.2 mm i.d. methyl phenyl silicone fused silica capillary column

702 703 704 705

Table 3: Average of percentage composition of the FAs of Acidovorax citrulli (AcM6, n = 2). Comparison of the present sample (AcM6 2), the sample cultivated half a year ago (AcM6 1) and exemplarily by means of two different haplotypes (A/ E) of Acidovorax citrulli, which were analysed by Walcott et al. (2000) [39]. FAME

AcM6 1 [%]

AcM6 2 [%]

variety 10:0 12:0 13:0 14:0 i15:0 a15:0 15:0 i16:0 16:0 17:0 18:0 14:1 (#2) 14:1n-5 (#3) 15:1n-5 (#2) 16:1n-9 (#1) 16:1n-7 (#2) 17:1 (#3) 17:1 (#4) 17:1n-8 (#5) 17:1 (#6) 18:1 (#3) 18:1n-9 (#4) 18:1n-7 (#5) 18:1 (#6) 3-OH-10:0 (#1) 3-OH-11:0 3-OH-12:1 3-OH-12:0 (#3) Instrument

20

16

Column

0.1 2.9

0.2 2.2

3.2 0.01 0.01 1.1 0.02 42.7 0.2 0.3

0.01 0.4 43.6 0.2 0.3 0.04 0.09 0.2 1.3 42.1 0.06 0.05 0.02 0.01 0.2 0.1 7.7 0.01 1.1

Acidovorax citrulli haplotype A/ E by Walcott et al. (2000) [39] [%] 13 0.8/ 0.7 3.0/ 3.6 1.7/ 1.8

2.0/ 1.7 31.3/ 28.4 0.3/ 2.2

- / 0.6 1.3 39.3

43.7/ 41.5

0.2 0.1 7.6

8.0/ 5.4

1.0

GC/MS 60 m x 0.25 mm i.d. 90% biscyanopropyl, 10% cyanopropylphenyl polysiloxane capillary column

706 707

35

6.7/ 8.4 0.3/ 0.4 2.4/ 4.2 1.0/ 3.2 Gas-liquid chromatography 30 m x 0.25 mm i.d. phenyl methyl silicone fused silica capillary column

708 709 710

Table 4: Average of percentage composition of the FAs of Pseudomonas syringae pv. tomato (Pst, n = 2). Comparison of the present sample and a Pst sample analysed by Stead (1992) [40]. FAME variety 10:0 12:0 a13:0 14:0 i16:0 16:0 i17:0 17:0 18:0 16:1n-7 (#2) 18:1 (#2) 18:1n-9 (#4) 18:1n-7 (#5) 3-OH-10:0 (#1) 2-OH-12:0 3-OH-12:0 (#3) Instrument Column

Pst [%] 14 0.07 5.02 0.03 0.37 0.02 33.2

Pst sample by Stead (1992) [40] [%] 10 trace 4.7 (± 0.3) 0.2 (± 0.1) 26.0 (± 1.4) trace

0.07 1.95 41.9 0.93 0.15 14.4 1.05 0.78 GC/MS 60 m x 0.25 mm i.d. 90% biscyanopropyl, 10% cyanopropylphenyl polysiloxane capillary column

711 712 713 714 715

36

40.5 (± 1.8) 17.8 (± 1.1) 3.0 (± 0.4) 2.6 (± 0.1) 4.0 (± 0.2) GC/FID 25 m methyl silicone fused silica capillary column

716 717

Table 5: Average of percentage composition of the FAs of Xanthomonas perforans (Xp, n = 2) of the present study. FAME variety 10:0 i11:0 a11:0 i12:0 12:0 i13:0 a13:0 13:0 i14:0 14:0 i15:0 a15:0 15:0 i16:0 16:0 i17:0 a17:0 17:0 18:0 16:1n-9 (#1) 16:1n-7 (#2) i17:1 (#1) 17:1n-8 (#5) 17:1 (#6) 18:1n-9 (#4) 18:1n-7 (#5) 3-OH-i11:0 (#2) 3-OH-12:0 (#3) 3-OH-i13:0 (#4) Instrument Column

Xp [%] 29 0.5 2.4 0.2 0.5 0.4 3.9 1.1 0.1 1.7 2.5 19.1 11.2 3.1 3.4 14.8 10.3 2.1 1.5 0.4 1.4 13.8 1.7 1.4 0.3 0.8 0.7 0.5 0.2 0.2 GC/MS 60 m x 0.25 mm i.d. 90% biscyanopropyl, 10% cyanopropylphenyl polysiloxane capillary column

718 719

37

720 721 722

Table 6: Average of percentage composition of the FAs of Clavibacter michiganensis subsp. michiganensis (Cmm, n = 2). Comparison of the present sample and of 45 reference strains analysed by Gitaitis and Beaver [41]. FAME variety 10:0 i11:0 a11:0 12:0 saturated i13:0 a13:0 13:0 i14:0 14:0 i15:0 a15:0 15:0 i16:0 16:0 i17:0 a17:0 17:0 18:0 i19:0 a15:1 (#1) 16:1n-9 (#1) 16:1n-7 (#2) a17:1 (#2) 17:1 (#4) 17:1 (#7) 18:1n-9 (#4) 18:1n-7 (#5) monoenoic (#1) monoenoic (#2) 3-OH-i11:0 (#2) 3-OH-i13:0 (#4) Instrument Column

Cmm of 45 reference strains [41] [%]

Cmm [%] 31

7 0.7 5.4 0.1 1.8 (± 3.2) 0.1 0.4 0.1 0.02 0.7 4.0 37.8 11.3 1.7 1.2 16.0 5.0 0.4 0.3 0.3 0.2 1.0 2.8 1.4 2.2 3.8 1.3 0.8 0.3 0.2 0.2 0.5 0.1

GC/MS 60 m x 0.25 mm i.d. 90% biscyanopropyl, 10% cyanopropylphenyl polysiloxane capillary column

723 724

38

0.9 (± 1.2) 40.9 (± 13.8) 13.9 (± 7.0) 3.7 (± 3.5) 21.3 (± 10.0)

8.5 (± 13.2)

Gas-liquid chromatography 30 m x 0.25 mm i.d. phenyl methyl silicone fused silica capillary column

725 726 727 728

Table 7: Average of percentage composition of the FAs of Streptomyces scabies (Ssc, n = 2). Comparison of the present sample and of pathogenic and scabsuppressive Ssc analysed by Ndowora et al. 1996 [43] and of two different groups of Ssc analysed by Paradis et al. (1994) [44]. FAME

Ssc [%]

variety i13:0 i14:0 14:0 i15:0 a15:0 15:0 i16:0 16:0 i17:0 a17:0 17:0 i18:0 15:1 i16:1 16:1n-7 (#2) 9-methyl-16:0 i17:1 (#1) a17:1 (#2) 17:1 (#3) 17:1 (#4) 17:1n-8 (#5) 17:1 (#7) 18:1 (#1) Instrument

18

Column

3.4 0.2 8.1 26.1 1.2 22.2 7.9 6.0 16.0 0.5 0.1

Ssc pathogenic/ scabsuppressive by Ndowora et al. (1996) [43] [%] 16 - / 0.3 11.0/ 7.6 9.2/ 13.5 11.0/ 21.5 5.3/ 3.9 27.6/ 25.3 5.1/ 2.3 2.0/ 2.1 4.6/ 6.5 0.5/ 0.8/ 0.9 7.3/ 3.2 6.9/ 4.2 3.7/ 3.4

0.6 1.4 1.4 0.9 3.4

2.3/ 3.8

Ssc group 1/ group 2 A by Paradis et al. (1994) [44] [%] 13 1.33/ < 1 3.13/ 2.17 4.59/ 1.03 11.20/ 17.46 15.30/ 23.77 2.55/ 1.31 7.33/ 10.79 30.78/ 15.01 2.68/ 8.18 4.58/ 10.80

12.28/ 3.64 1.63/ 2.66 < 1/ 1.57

1.5/ 0.7 0.3 1.8 GC/MS 60 m x 0.25 mm i.d. 90% biscyanopropyl, 10% cyanopropylphenyl polysiloxane capillary column

GC/FID

GC/FID

25 m x 0.2 mm i.d. 5% phenyl methyl silicone fused silica capillary column

25 m x 0.2 mm i.d. fused silica column

729 730

39

731

Highlights

732 733



GC/MS methods for fatty acids in cultures of six relevant pathogenic bacteria.

734 735



44 saturated, branched chain, monoenoic and hydroxy fatty acids could be detected.

736 737 738



Methods allowed to differentiate between 2- (1) and 3-OH-fatty acids (18 detected).

739 740 741



The fatty acid pattern of Clavibacter michiganensis differed from literature data.

742 743



The fatty acid pattern of Xanthomonas perforans was presented the first time.

744 745

40

746

CRediT author statement

747 748 749

Nina Wiedmaier-Czerny: Investigation, Formal analysis, Visualization, Writing – Original draft.

750

Dorothee Schroth: Investigation.

751

Shiri Topman: Investigation, Writing - review & editing.

752

Aya Brill: Investigation.

753

Saul Burdman: Methodology; Resources; Supervision; Writing - review & editing.

754

Zvi Hayouka: Conceptualization; Funding acquisition; Investigation; Supervision; Writing -

755

review & editing.

756

Walter

757

Supervision; Writing - review & editing.

Vetter:

Conceptualization;

Funding

758 759 760

41

acquisition;

Investigation;

Methodology;

761

Declaration of interests

762 763 764

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

765 766 767 768

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

769 770 771 772 773

42