Volatile organic compounds produced by the phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria 85-10

Article abstract of DOI:10.3762/bjoc.8.65

Xanthomonas campestris is a phytopathogenic bacterium and causes many diseases of agricultural relevance. Volatiles were shown to be important in inter- and intraorganismic attraction and defense reactions. Recently it became apparent that also bacteria emit a plethora of volatiles, which influence other organisms such as invertebrates, plants and fungi. As a first step to study volatile-based bacterial-plant interactions, the emission profile of Xanthomonas c. pv. vesicatoria 85-10 was determined by using GC/MS and PTR-MS techniques. More than 50 compounds were emitted by this species, the majority comprising ketones and methylketones. The structure of the dominant compound, 10-methylundecan-2-one, was assigned on the basis of its analytical data, obtained by GC/MS and verified by comparison of these data with those of a synthetic reference sample. Application of commercially available decan-2-one, undecan-2-one, dodecan-2-one, and the newly synthesized 10-methylundecan-2-one in bi-partite Petri dish bioassays revealed growth promotions in low quantities (0.01 to 10 ?mol), whereas decan-2-one at 100 ?mol caused growth inhibitions of the fungus Rhizoctonia solani. Volatile emission profiles of the bacteria were different for growth on media (nutrient broth) with or without glucose.

Full text of DOI:10.3762/bjoc.8.65

Volatile organic compounds produced
by the phytopathogenic bacterium
Xanthomonas campestris pv. vesicatoria 85-10
Teresa Weise1, Marco Kai1,2, Anja Gummesson3, Armin Troeger4,
Stephan von Reuß4,5, Silvia Piepenborn1, Francine Kosterka1,
Martin Sklorz3, Ralf Zimmermann3, Wittko Francke4 and Birgit Piechulla*1,§
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 579–596.
1University of Rostock, Institute of Biological Sciences,
doi:10.3762/bjoc.8.65
Albert-Einstein-Str. 3, 18059 Rostock, Germany, 2present address:
Max-Planck Institute for Chemical Ecology, Hans-Knoell-Str. 8, 07745
Jena, Germany, 3Joint Mass Spectrometry Centre of the University of
Rostock, Chair of Analytical Chemistry, Albert-Einstein-Str. 1, 18059
Rostock, Germany and the Cooperation group „Comprehensive
Molecular Profiling“, Helmholtz Zentrum München, Ingolstädter
Landstraße 1, 85764 Oberschleißheim, Germany, 4University of
Hamburg, Institute of Organic Chemistry, Martin-Luther-King-Platz 6,
20146 Hamburg, Germany and 5present address: Boyce Thompson
Institute, Cornell University, 1 Tower Road, Ithaca, NY, 14853, USA
Received: 09 February 2012
Accepted: 19 March 2012
Published: 17 April 2012
This article is part of the Thematic Series "Biosynthesis and function of
secondary metabolites".
Guest Editor: J. S. Dickschat
© 2012 Weise et al; licensee Beilstein-Institut.
License and terms: see end of document.
Email:
Birgit Piechulla* - birgit.piechulla@uni-rostock.de
* Corresponding author
§ Tel: 0049 381 4986130; Fax: 0049 381 4986132
Keywords:
Aspergillus nidulans; Fusarium solani; growth inhibition and
promotion; methylketones; 10-methylundecan-2-one; Rhizoctonia
solani; volatile organic compound (VOC); Xanthomonas campestris
pv. vesicatoria
Abstract
Xanthomonas campestris is a phytopathogenic bacterium and causes many diseases of agricultural relevance. Volatiles were shown
to be important in inter- and intraorganismic attraction and defense reactions. Recently it became apparent that also bacteria emit a
plethora of volatiles, which influence other organisms such as invertebrates, plants and fungi. As a first step to study volatile-based
bacterial–plant interactions, the emission profile of Xanthomonas c. pv. vesicatoria 85-10 was determined by using GC/MS and
PTR–MS techniques. More than 50 compounds were emitted by this species, the majority comprising ketones and methylketones.
The structure of the dominant compound, 10-methylundecan-2-one, was assigned on the basis of its analytical data, obtained by
GC/MS and verified by comparison of these data with those of a synthetic reference sample. Application of commercially available
decan-2-one, undecan-2-one, dodecan-2-one, and the newly synthesized 10-methylundecan-2-one in bi-partite Petri dish bioassays
579

Beilstein J. Org. Chem. 2012, 8, 579–596.
revealed growth promotions in low quantities (0.01 to 10 μmol), whereas decan-2-one at 100 μmol caused growth inhibitions of the
fungus Rhizoctonia solani. Volatile emission profiles of the bacteria were different for growth on media (nutrient broth) with or
without glucose.
Introduction
Plant surfaces are inhabited by diverse and complex communi- 19]. Recently, a new field of interest emerged, when it became
ties of microorganisms, although these habitats may be hostile apparent that also bacteria emit a plethora of volatiles, which
environments, e.g. [1-6]. Bacteria are the most dominant inhabi- may influence other organisms, such as invertebrates, plants and
tants, e.g., more than 107 cells per cm2 of leaf surface are fungi [20,21]. There is increasing information about those
present, but also filamentous fungi and yeasts are found [7,8]. volatiles emitted by bacteria, especially rhizobacteria, e.g., [22-
One of the most abundant bacterial genera in the phyllosphere is 25], which influence the growth of fungi [22,23,26,27]. In most
Xanthomonas; X. campestris is the dominant species, and at cases the bacterial volatiles showed inhibitory activities
least 141 pathovars invasive against several plant species are (reviewed in [20]). We, therefore, hypothesized that volatile
known, including many of agricultural relevance [9]. Promi- compounds emitted by Xanthomonas campestris pv. vesica-
nent and widespread diseases caused by Xanthomonas species toria 85-10 may also play a role as antagonistic weapons
include diseases such as bacterial spot on peppers and tomatoes, against competitive fungi. As a first approach in our investi-
citrus canker, and bacterial blight disease in rice [9,10]. Unrav- gations, we analysed the profiles of its volatiles. Only two
elling the mechanisms for phytopathogenicity and virulence of volatile organic compounds released by X. campestris have so
X. campestris resulted in the identification of a type-three secre- far been described: 11-methyldodec-2Z-enoic acid [13] and
tion system through which bacterial effector proteins enter the γ-butyrolactone [28]. To extend this preliminary information we
plant cells to interfere with cellular processes, to the benefit of carried out GC/MS and proton transfer reaction (PTR)–MS
the bacterium [11]. However, some plants react to the effector analyses with X. c. pv. vesicatoria 85-10. In addition, we aimed
proteins by local cell death, a hypersensitive response, and are at structure elucidation and on temporal variation of profiles of
able to escape bacterial spreading and invasion. Apart from volatiles produced upon growth on different media.
these cell-to-cell-based interactions, additional modes of action
Results and Discussion
of X. campestris may influence growth of the plant directly or
Effect of Xanthomonas campestris pv. vesi-
catoria volatiles on fungal growth
indirectly. Although antagonistic features of Xanthomonas spp.
against fungi have been only sparsely studied, it is known that
Xanthomonas competes with fungi [3]. The bacteria take up
nutrients more rapidly and in larger amounts as compared to the
germ tubes of fungi. This could be advantageous for
Xanthomonas during competition for nutrients. Furthermore,
some antagonistic activity of two Xanthomonas isolates against
Verticillium dahliae was documented [12]. Since only a little or
no production of lytic enzymes or siderophores could be
observed, other mechanisms must exist that promote bacterial
growth versus fungal growth. It was shown that 11-methyl-
dodec-2Z-enoic acid, known as quorum-sensing signal,
produced by X. campestris pv. campestris repressed hyphal
development in Candida albicans [13], and Hogan et al. [14]
concluded that either the bacteria modulate the fungal behavior
or that C. albicans (or related fungi) responded to the presence
of antagonistic bacteria in such a way that it was advantageous
for the bacteria.
So far neglected in former investigations was the possibility that
volatiles emitted from X. c. pv. vesicatoria 85-10 may influ-
ence plant growth directly or indirectly, e.g., through fungi. As
a first step to investigate whether Xanthomonas volatiles effect
other organisms, three fungi, Aspergillus nidulans, Fusarium
solani and Rhizoctonia solani, were cocultivated with X. c. pv.
vesicatoria 85-10 in compartmentalized Petri dishes (Figure 1).
This ensures that only volatiles can diffuse between the
compartments to act on the fungal test organisms. Since it is
known that growth media influence the pattern of bacterial
volatiles [29], nutrient broth with and without the addition of
glucose, NBG and NB, respectively, were investigated.
Figure 1A–D demonstrates the effects of Xanthomonas volatiles
on R. solani in such a culture system. In the absence of bacteria
the fungus exhibits circular mycelium growth (control,
Figure 1A and Figure 1C), while growth was retarded when X.
c. pv. vesicatoria 85-10 was growing in the other compartment
(Figure 1B and Figure 1D). Growth of R. solani was more
inhibited when X. c. pv. vesicatoria 85-10 grew on NB
compared to less inhibition when it was grown on NBG (98%
vs 55%, Figure 1B and Figure 1D, respectively). The same ten-
dency was observed for the growth of A. nidulans and F. solani.
Volatiles were also shown to support and facilitate cross-
kingdom interactions, such as plant–insect communications as
bi- and tritrophic attractions and defenses [15]. For example,
volatiles emitted by vegetative plant tissues suppress the growth
of microorganisms, both in the case of bacteria and fungi [16-
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Beilstein J. Org. Chem. 2012, 8, 579–596.
Figure 1: Cocultivation of Xanthomonas campestris pv. vesicatoria 85-10 with three fungi on different media. (A) Control experiment: Cultivation of
Rhizoctonia solani on NB (day 4). (B) Cocultivation of X. c. pv. vesicatoria 85-10 on NB with Rhizoctonia solani (day 4). (C) Control experiment: Culti-
vation of Rhizoctonia solani on NBG (day 4). (D) Cocultivation of X. c. pv. vesicatoria 85-10 on NBG with Rhizoctonia solani (day 4). (E) Quantifica-
tion of the inhibition of fungal mycelium growth during cocultivation with X. c. pv. vesicatoria 85-10, either grown on NB (black column) or NBG (grey
column). Fungi: Aspergillus nidulans, Fusarium solani and Rhizoctonia solani. (4 dpi: four days after inoculation; NBG: nutrient broth (NB) II agar plus
1.1% glucose; *: growth significantly different compared to the control, p < 0.01 according to t-test).
When X. c. pv. vesicatoria 85-10 grew on NB, A. nidulans was specific reactions. Similar results were obtained when ten
inhibited by 85% and F. solani by 14%, while the inhibition of different rhizobacteria were tested with various fungi [27]. Most
both fungi on NBG was only 11% and 3.5%, respectively. The of the tested fungi, including R. solani, Verticillium dahliae,
inhibitory potential of X. c. pv. vesicatoria 85-10 volatiles on Paecilomyces carneus and Sclerotinia sclerotiorum were
fungi was less pronounced when the bacteria grew on glucose- strongly inhibited by the rhizobacteria (Serratia spp.,
containing media (Figure 1E), indicating that growth suppres- Pseudomonas spp., Stenotrophomonas spp.), and only F. solani
sion depended on the supplied nutrient source. Fiddaman and appeared to be resistant against the bacterial volatiles. Also
Rossall [29] described similar results, since the addition of Muscodor albus volatiles only partially influence the growth of
D-glucose but not L-glucose lead to the formation of inhibitory F. solani, while other fungi are much more severely inhibited
volatiles by Bacillus subtilis. The milder inhibitory potential of [30]. Bacterial volatiles may also directly influence plant
X. c. pv. vesicatoria 85-10 growing on NBG, may be explained growth. Arabidopsis thaliana and Physcomitrella patens were
by the suggestions that (i) inhibitory volatiles were produced in exposed to mixtures of volatiles emitted by various bacteria.
higher amounts when grown on NB medium, (ii) inhibitory Depending on the bacterial species or experimental setup, either
volatiles were only produced in the peptone-rich NB medium, promotion or inhibition of plant growth was observed
(iii) glucose suppressed the production of inhibitory volatiles, [25,27,31-35]. Individual compounds such as acetoin and 2,3-
for example through catabolite repression, or (iv) the emission butanediol acted as plant-growth-promoting agents [31], while
of inhibitory volatiles was delayed.
dimethyl disulfide and 2-phenylethanol inhibited plant growth
[34,36]. The inhibitory potential of volatiles of different
Interestingly, Fusarium solani was only weakly influenced by Xanthomonas campestris species/isolates on Caenorhabditis
the volatiles of X. c. pv. vesicatoria 85-10, indicating species- elegans and on bacteria was also shown [20,37].
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Beilstein J. Org. Chem. 2012, 8, 579–596.
Volatile emissions of Xanthomonas
identical mass spectrum. As both compounds eluted between
undecan-2-one (31) and dodecan-2-one (38) (small amounts of
which were found to be present among the natural volatiles),
retention indices with methylketones as reference standards
campestris pv. vesicatoria 85-10
GC/MS analysis of volatiles released by X. c. pv.
vesicatoria 85-10
The volatiles emitted by X. c. pv. vesicatoria 85-10 showed were calculated. Corresponding data published for methyl-
strong effects on the tested fungi (Figure 1). Therefore, it was branched hydrocarbons and increment calculations strongly
interesting to carry out investigations on the qualitative and suggested iso-branching for the main compound and anteiso-
quantitative profiles of volatiles produced by X. c. pv. vesica- branching for the later-eluting isomer [38-40]. In fact, mass
toria 85-10. Samples of volatiles were obtained by growing the spectra and retention times (co-injection) of 10-methylundecan-
bacteria in 10 L of liquid medium and trapping the volatiles on 2-one (34) and 9-methylundecan-2-one (35) synthesized in our
SuperQ. Upon application of GC/MS, more than 50 com- lab (Scheme 1) matched those of the respective natural prod-
pounds were identified among a large number of components ucts. Small amounts of the corresponding alcohols, 10-methyl-
present in only very small amounts (Figure 2, Figure 3, undecan-2-ol (36) and 9-methylundecan-2-ol (37) (stereochem-
Table 1). Contaminants (see Table 1) are marked in Figure 2. istry not determined) were also detected by GC/MS and
The scent released by X. c. pv. vesicatoria 85-10 was shown to co-injection. As the headspace volatiles contained a continuous
be particularly rich in ketones and corresponding alcohols. In row of homologous straight-chain methylketones, identification
addition to straight-chain methylketones from hexan-2-one of additional compounds with similar structures was largely
(compound 1 in Figure 3 and Table 1) to pentadecan-2-one facilitated: the unbranched ketones were found to be always
(50), several branched-chain methylketones could be identified accompanied by the iso-branched isomers and in some cases
(Figure 3, Table 1). The volatiles were found to be largely also by the anteiso-branched ones. Corresponding methyl-
dominated by a substance (Figure 2, compound 34) showing a carbinols could also be detected. The position of the double
mass spectrum that was very similar to that of dodecan-2-one. bond in a pentadecen-2-one (49), a member of the identified
A slightly later-eluting minor component furnished an almost suite of methylketones, remained unassigned.
Figure 2: GC/MS-chromatogram (total ion current) of the headspace of X. c. pv. vesicatoria 85-10 grown in 10 L liquid nutrient broth without glucose.
Compound labels are the same throughout this paper.
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Beilstein J. Org. Chem. 2012, 8, 579–596.
Figure 3: Structures of compounds emitted by Xanthomonas campestris pv. vesicatoria 85-10. Compound labels are the same throughout this paper.
Table 1: GC/MS analysis of Super-Q trapped volatiles emitted by Xanthomonas campestris pv. vesicatoria 85-10 grown in 10 L nutrient broth without
glucose. Compound labels are the same throughout this paper.
compound
tR [min]
retention index I
rel. area [%]
compound
1
8.23
778
0.7
1.6
2.5
tr
hexan-2-one
2
8.96
795
2-methylpropyl acetate
n-octane
3
9.10
798
4
10.50
11.28
11.40
11.67
12.36
12.53
12.92
14.36
14.78
15.14
15.26
15.88
16.22
16.97
17.37
18.51
18.71
18.71
20.29
20.85
23.13
23.37
23.69
23.99
830
5-methylhexan-2-one
2-methylbutyl acetate
3-methylbutyl acetate
heptan-2-one
5
848
5.4
2.4
0.2
0.4
1.4
2.0
0.5
tr
6
851
7
857
8
873
2,5-dimethylpyrazine
2-methylpropyl propionate
n-nonane
9
877
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
886
919
6-methylheptan-2-one
5-methylheptan-2-one
2-methylbutyl propionate
3-methylbutyl propionate
octan-2-one
929
938
3.9
0.7
tr
940
955
963
0.9
1.3
2.5
4.0
3.7
tr
2,3,5-trimethylpyrazine
hexyl acetate
981
990
benzylalcohol
1018
1022
1022
1061
1074
1131
1137
1145
1152
3-methylbutyl 3-methylbutyrate
acetophenone
7-methyloctan-2-one
nonan-2-one
5.6
8.4
52.8
11.6
1.4
tr
2-phenylethanol
8-methylnonan-2-one
7-methylnonan-2-one
8-methylnonan-2-ol
7-methylnonan-2-ol
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Table 1: GC/MS analysis of Super-Q trapped volatiles emitted by Xanthomonas campestris pv. vesicatoria 85-10 grown in 10 L nutrient broth without
glucose. Compound labels are the same throughout this paper. (continued)
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
24.60
26.82
27.31
28.73
29.23
29.56
31.32
31.58
31.76
32.02
32.66
34.68
35.08
36.37
36.76
38.64
38.89
39.03
39.27
39.87
42.02
42.45
43.19
45.24
45.48
49.69
1168
1224
1237
1274
1287
1296
1344
1351
1356
1363
1380
1437
1448
1485
1496
1552
1559
1563
1570
1589
1655
1668
1691
1757
1765
1907
2.9
decan-2-one
5.5
2-phenylethyl acetate
9-methyldecan-2-one
undecan-2-one
4.9
19.7
1.4
undecan-2-ol
3.7
3,6-dimethyl-2-(3-methylbutyl)pyrazine
10-methylundecan-2-one
9-methylundecan-2-one
10-methylundecan-2-ol
9-methyundecan-2-ol
dodecan-2-one
100.0
28.3
4.9
4.0
1.4
25.0
1.7
geranylacetone
11-methyldodecan-2-one
tridecan-2-one
17.5
tr
tridecan-2-ol
15.7
8.8
12-methyltridecan-2-one
11-methyltridecan-2-one
12-methyltridecan-2-ol
11-methyltridecan-2-ol
tetradecan-2-ol
3.0
0.7
1.4
0.6
13-methyltetradecan-2-one
pentadecen-2-one
17.7
2.1
pentadecan-2-one
0.5
14-methylpentadecan-2-one
13-methylpentadecan-2-one
terpenoid?
0.4
29.8
contaminants, also in blank
a
b
c
d
e
17.76
23.01
30.97
31.19
36.79
1000
1128
1334
1340
1497
5.1
2-ethylhexanol
8.8
2-ethylhexyl acetate
hydrocarbon
17.8
20.5
6.7
hydrocarbon
2,4-bis-tert-butylphenol
Some of the identified ketones released by X. c. pv. vesicatoria
85-10 were known to be emitted by other bacteria, e.g.,
undecan-2-one (31) was emitted from Serratia sp. 2675,
Pseudomanas fluorescens, P. chlororaphis, P. aurantiaca and
P. corrugata [22,41]. Dodecan-2-one (38) was found among the
volatiles of Serratia strains [41], and decan-2-one (28) was
released by an epilithic cyanobacterial biofilm of Rivularia sp.
and Calothrix parietina [42]. The main component among the
volatile compounds released by X. c. pv. vesicatoria 85-10,
namely 10-methylundecan-2-one (34), has been reported from
plants [43-46] and bacteria, e.g., from the myxobacterium Stig-
matella aurantiaca and arctic bacteria of the cytophaga-
flavobacterium-bacteroides group [47,48]. In some of these
bacteria a pattern of methylketones similar to that of X. c. pv.
vesicatoria 85-10 was found: A continuous row of homologous
straight-chain methylketones accompanied by iso-branched
isomers, showing 10-methylundecan-2-one (34) as a major
Scheme 1: Synthesis of 10-methylundecan-2-one (34) and 9-methyl-
undecan-2-one (35).
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Beilstein J. Org. Chem. 2012, 8, 579–596.
component. To the best of our knowledge, the isomeric quently, the anteiso-branched isomers of compounds 21, 30, 40,
9-methylundecan-2-one (35) has not been identified from a and 48 (Table 1) were absent among the natural volatiles.
natural source before.
It is interesting to note that 10-methylundecan-2-one (34), the
Possible biosynthesis of methylketones found in
main component among the less volatile compounds emitted by
Xanthomonas campestris pv. vesicatoria 85-10
X. c. pv. vesicatoria 85-10, shows a close biogenetic relation to
While odd-numbered, unbranched methylketones are clearly 11-methyldodec-2Z-enoic acid (compound V in Scheme 2),
biosynthesized from straight-chain even-numbered fatty acids which has been described earlier as a metabolite produced by
originating from the acetate pool, the biosynthesis of even- Xanthomonas [13]. Possible biogenetic pathways to the two
numbered congeners may start from propionate, a C3-unit. compounds are shown in Scheme 2. Chain elongation of
Consequently, odd-numbered fatty acids will yield even- 3-methylbutyryl-SCoA (I), produced from leucine, will give
numbered straight-chain methylketones. Leucine or valine may rise to the formation of (ω−1)-methylcarboxylic acids [47,50],
serve as the starters for the biosyntheses of iso-branched com- and 10-methylundecan-2-one (34) may be either a degradation
pounds [47-49], which, in this case, is supported by the pres- product of a longer branched-chain carboxylic acid or formed
ence of three esters of 3-methylbutanol and one of 2-methylbu- during an anabolic process. In any case, 11-methyl-3-oxode-
tanol. Similarly, iso-leucine (its involvement in biosynthesis canoyl-SCoA (III) will be the key compound in the formation
being supported by the presence of esters of 2-methylbutanol) of 34. Starting from I, three complete cycles of chain elonga-
will give rise to anteiso-branching. However, due to the general tion with malonyl-SCoA produces 9-methyldecanoyl-SCoA
principles of chain elongation with acetate/malonate units, in X. (II), which will yield III upon condensation with another
c. pv. vesicatoria 85-10 the corresponding methylketones malonyl-SCoA. After hydrolysis and decarboxylation, III will
showed an odd number of carbons along the chain. Conse- directly form 34. On the other hand, reduction of III would
Scheme 2: Suggested biosynthesis of methylketones found in Xanthomonas campestris pv. vesicatoria 85-10.
585

Beilstein J. Org. Chem. 2012, 8, 579–596.
furnish IV, and subsequent elimination of water would lead to 41, 43, 57, 71, 73 and higher masses such as 157, 171 and 185
V, the immediate precursor of 11-methyldodec-2Z-enoic acid. (Figure 4). As PTR–MS only provides molecular mass informa-
However, V may also be formed upon dehydrogenation of tion, an unambiguous structure elucidation is impossible.
11-methyldodecanoyl-SCoA (VI), which, in turn, may be However, the tentative assignments of some volatiles are
produced by chain shortening of 13-methyltetradecanoyl-SCoA. strongly supported by the results obtained during GC/MS
Further steps during β-oxidation will afford III via IV.
analysis (Figure 2, Table 1). The intensive signal at m/z 157
may be attributed to the sum of decan-2-one (28), 7-methyl-
With the exception of 8-methylnonan-2-one (24), an important nonan-2-one (25) and 8-methylnonan-2-one (24); that at m/z
component of the female-produced sex pheromone of the desert 171 to the sum of undecan-2-one (31) and 9-methyldecan-2-one
spider Agelenopsis aperta [51], no significant biological activi- (30); and that at m/z 185 to the sum of dodecan-2-one (38),
ties have yet been reported for the identified branched 9-methylundecan-2-one (35) and 10-methylundecan-2-one (34).
methylketones and methylcarbinols.
As the instrument is tuned to maximum sensitivity, even the
soft ionization by H3O+ could lead to some fragmentation due
to the relatively high setting of the extraction/drift-tube volt-
ages. Therefore, the m/z values of 41, 43, 57, 71, especially,
Various other volatiles are present in the profile of
X. c. pv. vesicatoria 85-10
In addition to these methylketones and methylcarbinols, trace may be fragments of short-chain alkylketones and aldehydes, as
amounts of esters, such as 3-methylbutyl acetate (6) and reported by Blake et al. in 2006 [54].
3-methylbutyl propionate (14), and the corresponding 2-methyl-
butyl esters 5 and 13 as well as 3-methylbutyl 3-methylbutyrate Changes in the compositions of headspace volatiles
(19) could be identified. Trace amounts of benzylalcohol (18), of Xanthomonas campestris pv. vesicatoria 85-10
acetophenone (20) and 2-phenylethanol (23) and its acetate (29) during growth on different media
along with the three alkylated pyrazines 8, 16, 33 were the only Different intensities of reactions of the three tested fungi were
aromatic components found in the bouquet, while geranylace- observed when X. c. pv. vesicatoria 85-10 was either grown on
tone (39) and a later-eluting yet unidentified compound, NB or NBG (Figure 1). To trace this phenomenon, we applied
showing a mass spectrum somewhat resembling that of solid-phase micro-extraction (SPME)–GC/MS as well as
farnesylacetone, represented terpenoid structures among the PTR–MS to monitor the patterns of headspace volatiles under
volatiles produced by X. c. pv. vesicatoria 85-10.
two different growth conditions as well as at two different time
points. The profiles of volatiles produced by X. c. pv. vesica-
toria 85-10 growing on agar composed of NB and NBG were
investigated at day 3 and day 6 after inoculation (Figure 5). A
PTR–MS of highly volatile compounds released by
X. c. pv. vesicatoria 85-10
In our previous studies it became evident that the analysis of complex pattern of volatiles could be detected when X. c. pv.
volatile profiles by GC/MS has its technical limitations for the vesicatoria 85-10 was grown on the NB medium (Figure 5A).
detection of highly volatile compounds [34,52]. To extend the The pattern did not change much between day 3 and day 6
spectrum of volatiles of X. c. pv. vesicatoria 85-10, proton (Figure 5A and Figure 5C, Table 2). The volatile profile of X. c.
transfer reaction mass spectrometry (PTR–MS) was addition- pv. vesicatoria 85-10 grown on the NBG medium (day 3)
ally used. The analyses of volatiles emitted by X. c. pv. vesica- proved to be simpler, both with respect to qualitative and quan-
toria 85-10 were performed after three days of incubation on titative composition, since only a few compounds could be
NB and NBG in Petri dishes (Figure 4A and Figure 4B, respect- traced (Figure 5B, Table 2). Interestingly, the number of
ively). At least 27 m/z values were found in the mass range detectable compounds increased after six days on NBG
between 30 and 250 with signals that were significantly (Figure 5D, Table 2). By comparison of the bouquets released
enhanced (5% confidence level) compared to those of the blank by X. c. pv. vesicatoria 85-10 growing on NB and NBG, only
control samples. If the proton affinity of an analyte molecule is three overlapping compounds above a threshold level of 2 × 106
higher than the one of H3O+ the proton transfer reaction is more were observed at day 3, whereas during continued incubation
or less quantitative (quasi-first-order reaction kinetics). Thus the the compositions of bacterial volatiles became more alike, since
detection efficiency of compounds is rather independent of the eight compounds appeared in both chromatograms (Figure 5E,
nature of the analyzed molecule [53] and the signal obtained by Table 2). This observation suggested a delay of volatile emis-
normalization of the measured count rate to the primary ion sion for X. c. pv. vesicatoria 85-10 grown on NBG. Two
count rate is roughly proportional to the concentration. The scenarios may explain this observation: (i) catabolite repression
headspace concentrations measured by online PTR–MS during by glucose inhibited the synthesis of certain volatiles; or (ii) the
the incubation experiments cover about four orders of magni- easily accessible glucose was catabolized before other compo-
tude. The most abundant m/z values (M + H)+ were 59, 30, 33, nents of the medium were utilized.
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Beilstein J. Org. Chem. 2012, 8, 579–596.
Figure 4: PTR–MS mass spectra of Xanthomonas campestris pv. vesicatoria 85-10 volatiles after three days of incubation. (A) PTR–MS profile scan
of headspace volatiles of X. c. pv. vesicatoria 85-10 growing in a Petri dish with NB. (B) PTR–MS profile scan of headspace volatiles of X. c. pv. vesi-
catoria 85-10 growing in a Petri dish with NBG. Spectra are normalized to the primary ion signal (H3O+) and are blank corrected (i.e., spectra
recorded from the respective media without bacteria are subtracted). Signals that are significantly influenced by the growth media (5% confidence
level) are labeled by *. Only significant signals (mean subtracted by standard deviation) higher than 10−6 are shown and a log-scale on the ordinate
axis is used. Note that missing signals therefore do not necessarily indicate zero concentration but may also be due to the variance of the sample or a
blank signal higher than the mean value.
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Beilstein J. Org. Chem. 2012, 8, 579–596.
Figure 5: GC/MS analysis of volatiles emitted by Xanthomonas campestris pv. vesicatoria 85-10 grown on different media. (A) GC/MS-profile of
headspace volatiles of X. c. pv. vesicatoria 85-10 grown on NB at day 3. (B) GC/MS-profile of headspace volatiles of X. c. pv. vesicatoria 85-10 grown
on NBG at day 3. (C) GC/MS-profile of headspace volatiles of X. c. pv. vesicatoria 85-10 grown on NB at day 6. (D) GC/MS-profile of headspace
volatiles of X. c. pv. vesicatoria 85-10 grown on NBG at day 6. (E) Registration of volatiles reaching a threshold level >1 × 105 when X. c. pv. vesica-
toria 85-10 grew on NB (left circle) and NBG (right circle) (day 3: left panel, day 6: right panel). (F) Relative contribution (%) of the six major volatiles
emitted by Xanthomonas campestris pv. vesicatoria 85-10 at day 3 and 6 grown on NB and NBG.
Table 2: GC/MS analysis of SPME trapped volatiles emitted from Xanthomonas campestris pv. vesicatoria 85-10 grown in a Petri dish on NB or
NBG.a
NB
NBG
compound
identification
day 3
day 6
day 3
day 6
x1
x2
24
25
26
27
x3
—
—
+
—
+
—
—
+
+
+
—
8-methylnonan-2-one
7-methylnonan-2-one
8-methylnonan-2-ol
7-methylnonan-2-ol
—
+++
++
+
+++
++
+
+++
+
—
—
—
—
+
+
—
+
—
+
+
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Beilstein J. Org. Chem. 2012, 8, 579–596.
Table 2: GC/MS analysis of SPME trapped volatiles emitted from Xanthomonas campestris pv. vesicatoria 85-10 grown in a Petri dish on NB or
NBG.a (continued)
28
30
x4
decan-2-one
++
+++
+
++
+++
+
—
+
+
++
—
+++
+
9-methyldecan-2-one
—
—
++
+
31
x5
undecan-2-one
+++
+
+++
+
—
x6
—
+
+
—
+
+
34
35
36
37
x7
10-methylundecan-2-one
++++
++++
++
++
+
++++
++++
+++
+
++++
+++
—
+
9-methylundecan-2-one
++
—
—
—
—
+
10-methylundecan-2-ol
9-methylundecan-2-ol
—
+
—
+
38
39
40
x8
dodecan-2-one
++
++
++
++
+
++
+
geranylacetone
+
11-methyldodecan-2-one
++
+
—
—
—
+
+
—
++
—
—
+
x9
—
—
—
++
+
x10
41
x11
43
44
x12
x13
x14
x15
x16
—
—
++
+
tridecan-2-one
+
—
—
—
—
—
—
—
—
—
+
12-methyltridecan-2-one
++
++
+
++
++
+
++
++
+
11-methyltridecan-2-one
—
—
—
—
—
+
+
+
+
+
+
+
+
+
+
+
+
ax1–x16: compounds not identified; + = 0–105 counts; ++ = 105–106 counts; +++ = 106–107 counts; ++++ = 107–108 counts.
We also determined the contribution (%) of each of the six lower when bacteria grew on NBG for three days (Figure 4B).
major volatiles during bacterial growth (Figure 5F). The domi- Furthermore, m/z = 30, 36, 43, 44, 59, 60, 67 and 109 appeared
nant compound, 10-methylundecan-2-one (34) comprised about at higher levels in NB compared to NBG. Conversely, when
70% under the four growth conditions, followed by 10–12% of bacteria were grown on NBG, the signals at m/z = 39, 41, 57,
9-methylundecan-2-one (35), about 5–7% of 8-methylnonan-2- 71, 79 as well as 89 and 91 were enhanced. When the incuba-
one (24), 4–6% undecan-2-one (31), and 1–2% 9-methyldecan- tion time on NBG was prolonged to six days, even the alka-
2-one (30) and 11-methyltridecan-2-one (44). All other nones contributed significantly, but still in about five-fold lower
compounds contributed less than 1% to the bouquets. It is amounts.
interesting to note, that the ratios of the six major compounds
did not significantly vary (except for undecan-2-one (31) at day Individual volatiles of Xanthomonas campestris pv.
3 on NBG), indicating that the same metabolic pathways were vesicatoria 85-10 marginally influence fungal growth
active and only larger quantities/fluxes of the volatiles were As demonstrated in Figure 1, X. c. pv. vesicatoria 85-10
emitted under the four different growth conditions.
strongly inhibited the growth of Rhizoctonia solani and
Aspergillus nidulans and to a certain extent that of Fusarium
Similarly, as performed with the GC/MS analysis, PTR–MS solani. The inhibition was stronger when X. c. pv. vesicatoria
volatile profiles where obtained when X. c. pv. vesicatoria was 85-10 was grown on NB as compared to NBG. Both methods,
grown on NB and NBG at day 3 and 6. The PTR–MS pattern of GC/MS and PTR–MS, indicated the emission of a multitude of
X. c. pv. vesicatoria grown on NB for three (Figure 4A) and six volatile compounds, including many ketones. Decan-2-one (28),
days were quite similar (data not shown). It is remarkable that, undecan-2-one (31), dodecan-2-one (38) and 10-methyl-
in comparison, the signals for the later-eluting alkanones, i.e., undecan-2-one (34) were identified as major products of X. c.
m/z 157, 171 and 185 especially, were absent or significantly pv. vesicatoria 85-10. Since the ketones were released in
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Beilstein J. Org. Chem. 2012, 8, 579–596.
different quantities from both media, we tested these ketones cally, and they were most likely not significantly responsible for
individually in different amounts or in combination, to find out inhibitory effects on the growth (Figure 1) of the fungi by X. c.
whether the identified ketones influence the fungal growth pv. vesicatoria volatiles. We concluded that either the experi-
(Figure 6). They were applied repetitively every 24 h at concen- mental design of the bioassay was not appropriate or other
trations of 0.01, 0.1, 1.0, 10 and 100 μmol in 50 μL pentane volatiles account for the inhibitory effects. In the literature,
(10-methylundecan-2-one (34) was applied at 0.01, 0.1 and contradictory results were found for undecan-2-one (31). It is
1 μmol in 50 μL pentane). The area of the fungal mycelium was likely to be involved in the inhibition of sapstain fungi [40],
determined after four days of cocultivation. Only at a concentra- while Sclerotinia sclerotiorum was not affected by 31 [22]. It
tion of 100 μmol decan-2-one (28) the growth of R. solani was must also be considered that it is not known whether the emitted
inhibited, by 30% (Figure 6), while the other ketones did not volatile blends change qualitatively and quantitatively during
significantly influence the development of R. solani at any of the growth of X. c. pv. vesicatoria 85-10 [34]. Hence, to find
the tested concentrations. Surprisingly, undecan-2-one (31) the inhibitory component(s) and the bioactive concentrations,
promoted the growth of R. solani by 10–15% at concentrations the emitted blends of volatiles must be investigated more
from 0.01 to 10 μmol in 50 μL and dodecan-2-one (38) at 0.01 comprehensively.
and 0.1 μmol in 50 μL. 10-Methylundecan-2-one (34) had no
effect on the growth of R. solani. An additional bioassay, In addition, inorganic compounds have to be considered,
performed with a mixture of the four synthetic compounds including hydrogen cyanide (HCN) and ammonia. HCN is
(decan-2-one (28), undecan-2-one (31), dodecan-2-one (38), produced, e.g., by Pseudomonas spp. [55] and, due to its
10-methylundecan-2-one (34)) in the ratios that were emitted by slightly higher proton affinity than water, it should be detectable
the bacteria growing in the Petri dish (6.7%:5.5%:87.2%:0.6%), down to a concentration lower than 100 ppb by PTR–MS
revealed 10% inhibition at 9 μmol in 50 μL and slight promo- [56,57]. Nevertheless in our experiments with X. c. pv. vesica-
tion (ca. 6%) at 0.09 μmol in 50 μL. These results indicate that toria 85-10 we did not detect any significant signal at m/z = 28
the individual ketones neither acted additively nor synergisti- corresponding to HCN. Ammonia emissions by X. vesicatoria
Figure 6: Testing synthetic volatiles on the growth of Rhizoctonia solani. Synthetic commercially available and chemically synthesized ketones were
dissolved in pentane and applied on a filter-paper disc in aliquots of 50 μL. The filter paper was placed in the opposite compartment to the R. solani in
a bi-partite Petri dish. Defined dilutions or mixtures were applied every 24 h. At day 4 the diameter of the mycelium was measured and compared to
control plates containing pentane. Positive values represent growth promotion and negative values growth inhibition of R. solani compared to the
control. Data are from two experiments each replicated three times; the standard deviation is presented. (A) Decan-2-one (28) (black), undecan-2-one
(31) (light grey), dodecan-2-one (38) (white), and 10-methylundecan-2-one (34) (dark grey) were individually applied in 50 μL aliquots each day (day 0
to day 4). (B) A mixture of 28, 31, 38 and 34 (6.7%:5.5%:0.6%:87.2%) was applied in 50 μL aliquots every day (day 0 to day 4).
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Beilstein J. Org. Chem. 2012, 8, 579–596.
and of two Bacillus subtilis strains in cocultivation with and assignment of atomic compositions. Regarding quantitative
Neurospora crassa were described [58,59]. Ammonia is of analysis, some striking differences between results obtained
course an important nitrogen source for organisms, but recently with extracts or solid-phase micro extraction (SPME) applying
other roles were also attributed to ammonia. Nijland and conventional GC/MS and PTR–MS, respectively, have been
Burgess [60] suggested that ammonia acts as an olfactory cue in observed. As an example, in PTR–MS analyses the signal at
the communication of Bacillus licheniformis strains, and m/z = 109 is about as intensive as those produced by the methyl
Bernier et al. [61] showed that elevated ammonia levels lead to ketones at m/z = 157, m/z = 171, and m/z = 185 (Figure 4A). In
increased polyamine levels in E. coli with consequences for the contrast, in our GC/MS analyses the sum of 2,5-dimethyl-
membrane permeability and increased antibiotic resistance. pyrazine (8) and benzylalcohol (18) (Figure 2, Table 1), which
Other volatiles such as CO, CH4, CH2O and NO were not may give rise to m/z = 109 (provided that there is no abundant
detectable with the applied methods. However, the PTR compound that did not survive GC), never exceeded 5% of
provides evidence for the emission of sulfur-containing com- 10-methylundecan-2-one (34). These discrepancies require
pounds such as H2S (m/z = 35) and methanethiol (m/z = 49), as further investigations. The most important future analytical
has been suggested for other microorganisms [52]. The com- aspect will be online PTR–MS, tracing the dynamics in the
pound producing m/z = 33 was tentatively assigned to be emission of volatiles during incubation time, considering both
methanol in accordance with published data [52]. Methanol is qualitative and quantitative compositions of the profiles. In ad-
so far not known as a prominent compound released by dition, gas-chromatographic analyses of highly volatile com-
bacteria. In addition to the Enterobacteriaceae E.coli, Shigella pounds, by using either direct headspace injection or solid-
flexneri, and Salmonella enterica also X. vesicatoria may emit phase adsorption/thermodesorption separation on thick film
methanol. The release of CO2 due to the active tricarboxylic capillaries connected to a GC/MS system, will have to be
acid cycle is very likely, but CO2 does not accumulate at higher carried out.
than ambient concentrations in the open experimental system
[62] and, therefore, cannot be considered as a growth-promoting Conclusion
or -inhibitory component.
Profiles of volatiles emitted by Xanthomonas c. pv. vesicatoria
85-10 were investigated by using GC/MS and PTR–MS tech-
niques. More than 50 compounds were emitted by this species,
the majority comprising ketones and methylketones, including
Considerations concerning methodological
approaches
It is quite obvious that further research is necessary to identify 10-methylundecan-2-one (34) as the largely dominant compo-
the bioactive compounds, especially those sensitive and/or nent. The emission profiles differed depending on whether the
highly volatile substances that escaped conventional GC/MS- bacteria were grown on a medium with or without glucose. To
analysis. During our investigations on volatiles of Xanthomonas better understand the involvement of X. c. pv. vesicatoria
we noticed a discrepancy in the number of compounds detected volatiles in antagonistic processes against fungi, more eco-
in the headspace of a 10 L liquid-medium culture versus growth logical information is required. Most obviously it needs to be
on solid medium (Petri dish). Beside the known physiological addressed as to whether X. c. pv. vesicatoria 85-10 emits
adaptations of bacteria growing on liquid and solid media, the volatiles while growing in the phyllosphere and whether the
different trapping material (Super-Q versus polydimethyl- emission occurs continuously. Continuous volatile perception
siloxane) and the experimental procedure (30 hour trapping by the plant may be a prerequisite to induce signal cascades in
versus 2 hour trapping) most likely account for those differ- the plant, similarly to the behavior shown for repetitive
ences. As discussed above, the suitability of PTR–MS for com- wounding in contrast to single cuts in plants [65]. Furthermore,
pound identification is limited. Therefore, structure assign- a possible role of volatiles in achieving a better colonization of
ments are based on comparison with GC/MS data (i.e., as the aerial parts by X. c. pv. vesicatoria has to be considered.
performed also in this work) and literature-based information. Another question of concern is whether and how the plants are
Selectivity may be enhanced by using ammonia as the primary directly affected by the volatiles of X. c. pv. vesicatoria 85-10.
ion-source gas. Ammonia has a higher proton affinity than Plant growth promotion as well as inhibition is reflected at the
water, and therefore, some substance classes can be ionized molecular and physiological level of Arabidopsis thaliana and
selectively (e.g., nitrogen-containing compounds). Further of Physcomitrella patens [23,27,31,34,62,66]. However, in
information to discriminate nominally isobaric compounds most of these studies the bioactive compound(s) need to be
could be obtained by using proton-transfer-reaction ion-trap determined. Therefore, future tasks must focus on (i) the
mass spectrometry (PIT–MS), which allows tandem mass spec- comprehensive determination of volatile profiles of bacteria of
trometric experiments (MS/MS) [63,64], or by high-resolution the phyllosphere as well as in the rhizosphere and (ii) the identi-
PTR–TOF–MS for the determination of exact molecular masses fication of biologically active volatiles.
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Beilstein J. Org. Chem. 2012, 8, 579–596.
Experimental
Bacterial cultures
1 mL (OD600 = 0.8–1.4) was used as inoculum for a 250 mL
NB culture. The preculture (100 mL) was used to inoculate 10 L
Xanthomonas campestris pv. vesicatoria 85-10 was originally of the culture medium in a 20 L Duran® wide-necked glass
isolated from Capsicum sp. The bacterial strain was kindly flask closed with a specially designed glass cap (equipped with
provided by Prof. U. Bonas (University of Halle/Wittenberg). It an inlet and an outlet). During growth, the bacterial culture was
was stored in frozen stocks at −70 °C and grown on nutrient stirred with a magnetic stir bar at 600 rpm and incubated at
broth (NB) II agar (SIFIN, Berlin) either with or without 1.1% 30 °C. A diaphragm pump (Denver Gardner, Puchheim,
glucose (Merck, Darmstadt) at 30 °C.
Germany) pulled charcoal-purified, sterile air at a flow of
3 L/min through the system. Volatiles released during a
30 hours growth of X. c. pv. vesicatoria were trapped on Super-
Fungal isolates
Aspergillus nidulans (FGCS A4) was kindly provided by Q (100 mg, Alltech Associates, Deerfield, IL, USA). The
Dr. S. Busch (University of Göttingen). Fusarium solani and collected volatiles were eluted three times with 500 μL
Rhizoctonia solani KÜHN (RHI S0 WE) were obtained from dichloromethane and further analysed. Gas chromatography
the Strain Collection of Antagonistic Microorganisms (SCAM; coupled with mass spectrometry (GC/MS) was carried out by
University of Rostock, Microbiology). Fungal mycelium was linking a gas chromatograph HP 5890 (Hewlett Packard, Palo
grown on Sabouraud agar (Dinkelberg Analytics GmbH, Alto, US) to a double-focusing spectrometer (VG-7070/250SE,
Regensburg) with a mycelia plug from stock cultures stored at Vacuum Generators, Manchester, UK). By using helium as the
−70 °C. To maintain the fungi, every seventh day a mycelium carrier gas, separations were achieved with a 50 m × 0.25 mm
disk was transferred from the edge of an actively growing id CPSil5 fused silica column (Chrompack, Middleburg, The
fungus to a new agar plate. Cultures were incubated at 20 °C in Netherlands) under the following conditions: injector tempera-
the dark.
ture 250 °C, 1 min splitless, 1 min at 50 °C, then programmed
to increase to 290 °C at a rate of 3.5 °C/min. Identification of
volatiles was based on comparison of their mass spectra with
those reported in the literature [67,68] as well as with the
Cocultivation of Xanthomonas campestris pv.
vesicatoria 85-10 with fungi
Bacteria were grown on nutrient agar (NB II; peptone from analytical data of commercially available or specifically synthe-
casein 3.5 g·L−1, peptone from meat 2.5 g·L−1, peptone from sized reference compounds (co-injection). Synthetic 10-methyl-
gelatin 2.5 g·L−1, yeast extract 1.5 g·L−1, NaCl 5 g·L−1, agar- undecan-2-one and 9-methylundecan-2-one served as standards
agar 15 g·L−1, pH 7.2), and nutrient agar with glucose (NBG; for the structure assignments of other branched-chain methyl
NB plus 11 g·L−1 glucose) at 30 °C. Cocultivations of bacteria ketones on the basis of their mass spectra and retention indices.
and fungi were performed in bi-partite Petri dishes. One milli-
liter of the overnight cultures of Xanthomonas grown on NB or Cultivation on solid medium: In another analytical approach,
NBG was transferred into 100 mL fresh medium in 250 mL solid-phase micro extraction (SPME) was applied to trap
chicane flasks and incubated by shaking overnight at 30 °C. volatiles, which were subsequently analyzed by GC/MS.
Fifty microliters of these cultures were plated onto nutrient Bi-partite glass Petri dishes with a borehole (1 mm diameter) at
agar, with or without glucose, in one compartment of the Petri the side of one compartment were used for SPME. The precul-
dish and cultivated at 30 °C. Three days later (day 0 of coculti- ture (1 mL) was used to inoculate 100 mL NB or NBG medium
vation) a seven-day-old 6 mm disk of actively grown mycelium in a 250 mL chicane flask. After 24 h of incubation at 30 °C,
of the tested fungus was transferred to Sabouraud agar in the 50 μL was plated on NB or NBG agar on one side of the
other compartment of the Petri dish. Cocultivation was compartmentalized Petri dish. Similarly to the cocultivation
performed at 20 °C in the dark. Digital images were taken after tests, the inoculated plate was incubated for three days at 30 °C
four days. Growth of fungi was determined by measuring the under darkness. The collection of volatiles was performed at
diameter of the mycelium and compared to the control (without day 3 and day 6 after inoculation at 20 °C in the dark. A 100 μm
adjacent bacterial growth). Average values were calculated polydimethylsiloxane (PDMS; SUPELCO, Bellefonte, USA)
based on three experiments each with three to five replicates. coated SPME-fiber was preconditioned to remove all contami-
nants (30 min, 250 °C). Subsequently, the SPME needle was
Analysis of volatiles produced by
introduced into the Petri dish through the hole, and the fiber was
Xanthomonas campestris pv. vesicatoria
85-10
Cultivation in liquid medium: A single colony of X. c. pv.
vesicatoria was picked and suspended in 6 mL of NB medium.
After incubation for 24 h at 30 °C and shaking at 180 rpm,
extended into the headspace. The time for the adsorption and
accumulation of bacterial volatiles was set to 2 h. Volatile
compounds were thermally desorbed and analyzed by using a
GC/MS-QP5000 (Shimadzu; Kyoto, Japan, injection port set at
250 °C). The initial temperature of the DB5-MS column
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Beilstein J. Org. Chem. 2012, 8, 579–596.
(60 m × 0.25 mm × 0.25 μm; J&W Scientific, Folsom, Cali- step these normalized raw spectra were averaged and blank-
fornia, USA) was kept at 35 °C for 5 min, increased to 280 °C value corrected. Statistical analyses of bacterial metabolites on
at a rate of 10 °C/min, and then kept constant for 15 min. NB and NBG were performed by using a variant of the T-test,
Helium at a flow rate of 1.1 mL/min was used as the carrier gas with a 5% confidence level [71].
with a linear velocity of 28 cm/s. Electron ionization (EI) mass
spectra were taken at 70 eV. The mass range was m/z = 40–280. Syntheses of reference compounds
Compounds were identified by using available reference com- The syntheses of 10-methylundecan-2-one (34) and 9-methyl-
pounds and the library of the National Institute of Standards and undecan-2-one (35) proceeded straightforwardly, following a
Technology (NIST147) for the comparison of mass spectra, conventional alkyne approach (Scheme 1). Commercially avail-
retention times and Kovats indices [69]. Analysis of the volatile able (Aldrich) 4-pentyne-2-ol was reacted with benzyl chloride
emissions was replicated twice, and compounds emitted by the to yield the corresponding benzyl ether A. Subsequently, A was
NB or NBG agar (blank) were subtracted.
coupled to commercially available (Aldrich) 1-bromo-5-methyl-
hexane (B) according to the standard procedure [72]. The
Highly volatile components produced by X. campestris pv. vesi- resulting 2-benzyloxy-10-methylundec-4-yne (C) was hydro-
catoria 85-10 were monitored by proton transfer reaction mass genated over 10% Pd/C-catalyst at 1 atm. The crude secondary
spectrometry (PTR–MS). This technique allows continuous alcohol was oxidized with pyridinium dichromate [73,74] to
online monitoring of mixtures of volatiles at the parts-per- yield the corresponding ketone. Purification by column chroma-
billion (ppb) level. The method is a special variant of the well- tography on silica (ICN, pore size 60 Å, particle size 32–63 μm)
established chemical ionization mass spectrometry. In with cyclohexane/ethyl acetate 20:1 followed by Kugelrohr dis-
PTR–MS, the hydronium ion H3O+ is the primary species to tillation at 20 mmHg afforded 10-methylundecan-2-one (34) in
generate the protonated analyte (M + H)+, which is recorded by a purity of 98%. Analytical data of the product were identical to
the detector. The PTR–MS (Ionicon, Innsbruck, Austria) used those reported in the literature [47]. Following the same route as
in this investigation has been described elsewhere [70]. As a for the synthesis of 34 (Scheme 1), 9-methylundecan-2-one
modification to this original PTR–MS a heated inlet system was (35), was prepared by coupling 1-bromo-4-methylhexane (B′)
installed to prevent condensation and to enable also a qualita- [75] to A. Hydrogenation of the obtained C′ followed by oxi-
tive analysis of less volatile compounds. It consists of a deacti- dation of the obtained alcohol yielded 35.
vated 2 m long GC capillary (ID 0.53 mm, MXT Guard
Column, BGB-Analytik, Schlossboeckelheim, Germany) inte- Synthesis of 9-methylundecan-2-one (35): To a stirred, ice-
grated in a 1/8 inch copper tube and maintained at a tempera- cooled solution of 2.0 g (23.8 mmol) rac-4-pentyne-2-ol in
ture of 60 °C by using a heating hose (Horst GmbH, Lorsch, 50 mL dry THF was slowly added 1.20 g (49.9 mmol) sodium
Germany). The sample flow through the capillary is 80 mL/min. hydride. The reaction mixture was warmed to room tempera-
Drift tube pressure was set to 2.0 mbar and drift voltage to ture, and stirring was continued until the formation of hydrogen
600 V. These settings resulted in very sensitive measurements, ceased. Subsequently, a solution of 4.27 g (25.0 mmol) benzyl
but some side reactions led to fragmentation of, e.g., aldehydes, bromide in 50 mL dry THF was added slowly. After 3 h stir-
as described for a SWIFT instrument by Blake et al. [54]. Mass- ring at RT, 150 mL water was added, and the mixture was
to-charge ratios from 20 to 250 unified mass units (m/z) were extracted with three portions of diethyl ether at 50 mL each.
run by using a quadrupole at a dwell time of 100 ms per m/z The combined organic solutions were washed with a saturated
resulting in a repetition rate of about two full mass spectra per aqueous solution of sodium hydrogen carbonate and brine and
minute. Generally 100 spectra were recorded. For PTR-analysis dried over magnesium sulfate. The solvent was removed in
the Petri dish with growing bacteria were placed at day 3 and 6, vacuo, and the residue was submitted to flash chromatography
without the lid, into a glass compartment (Petri dish, 145 x (silica; cyclohexane/ethyl acetate 40:1) yielding 2.44 g
30 mm) with an in- and outlet. Charcoal-purified air was passed (29.8 mmol, 83%) of 2-benzyloxypent-3-yne (A).
over the culture and entered the PTR instrument through the
inlet. All analyses of bacterial metabolites were carried out in A solution of 2.70 g (15.5 mmol) of A in 50 mL dry THF was
triplicate. Blank samples of NB and NBG were analyzed twice cooled to −78 °C and deprotonated with 10.6 mL of a 1.6 M
to identify compounds directly related to outgasing of the solution of butyl lithium in hexane, which was added slowly.
nutrient agar. For data evaluation, raw spectra of each measure- After warming to RT, 3.05 g (17.0 mmol) of 1-bromo-4-methyl-
ment (100 spectra) were first normalized to the signal of the pri- hexane (B’), dissolved in 15 mL dry THF, was added dropwise.
mary ion (H3O+), to enable a semiquantitative data evaluation After heating under reflux for 12 h, the mixture was cooled to
and a direct comparison of spectra that were obtained at RT, and 150 mL of a saturated aqueous solution of ammonium
different days with a slightly different sensitivity. As a second chloride was added. After separation, the aqueous layer was
593

Beilstein J. Org. Chem. 2012, 8, 579–596.
extracted with three portions of diethyl ether at 50 mL each. (28:31:34:38 6.7%:5.5%:87.2%:0.6%) was tested as well. Fifty
The combined organic solutions were washed with brine and microliters of each mixture (0.09–9 μmol) were applied on filter
dried over magnesium sulfate. The solvent was removed in paper (1 cm2), which was deposited at the other compartment of
vacuo, and the residue was submitted to flash chromatography the Petri dish. Control experiments were performed with
(silica; cyclohexane/ethyl acetate 40:1) to yield 2.7 g pentane alone. Similarly to the cocultivation test, the bioassay
(10.0 mmol, 64%) of 2-benzyloxy-9-methylundec-4-yne (C′). A with individual compounds was performed at 20 °C in the dark.
solution of 2.53 g (9.3 mmol) of crude C′ in 50 mL methanol Every 24 h the filter paper was replaced by a freshly prepared
was hydrogenated overnight at 20 bar by using 20% PD/C-cata- filter paper with 50 μL of the respective test solution. Growth
lyst. The mixture was filtered over silica, and the solvent was inhibition of fungi was determined by measuring the diameter
removed in vacuo.
of the mycelium at day 4 of the experiment and by comparison
to the control experiment (pure pentane). Average values were
To a solution of 950 mg (5.1 mmol) of the crude 9-methylunde- calculated based on two repeat experiments each with three
cane-2-ol in 50 mL dichloromethane was added 2.3 g replicates.
(1.2 equiv) pyridinium dichromate, and the mixture was vigor-
ously stirred for 12 h at RT. As a gas-chromatographic control Acknowledgements
revealed the reaction to be incomplete, another 1.2 equiv of The authors thank the DFG for financial support (Pi153/26 resp.
pyridinium dichromate was added, and the mixture was heated FR507/19-1). We are grateful to Dr. Ulla Bonas for providing
under reflux for 6 h. After filtration over silica and concentra- Xcv 85-10 and Dr. S. Busch for Aspergillus nidulans
tion in vacuo, the crude ketone was purified by column chroma- (FGCS A4). We like to thank Claudia Dinse for technical assis-
tography on silica with cyclohexane/diethyl ether 20:1. A final tance.
Kugelrohr distillation of 20 mmHg afforded 855 mg (4.6 mmol)
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