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
2 3 4 5
Nina Wiedmaier-Czerny1, Dorothee Schroth1, Shiri Topman2, Aya Brill2, Saul Burdman3, Zvi Hayouka2* and Walter Vetter1*
6
1
7
Hohenheim, D-70593 Stuttgart, Germany
8
2
9
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
10
76100, Israel
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3
12
Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot
13
76100, Israel
Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of
14 15 16
* Corresponding authors:
17
Walter Vetter
18
Phone: +49 711 459 24016
19
Fax: +49 711 459 24377
20
E-Mail:
[email protected]
21 22
Zvi Hayouka
23
Phone: +97289489019
24
Fax: +97289489483
25
E-Mail:
[email protected]
26 27
1
28
Abstract
29
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
91
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