Correlation between lipophilicity of newly synthesized ionic liquids and selected: Fusarium genus growth rate

Article abstract of DOI:10.1039/c9ra02521a

The purpose of the present study was to examine the effectiveness of 23 different synthesized ionic liquids (ILs) on Fusarium culmorum and Fusarium oxysporum growth rate. The strategy of IL synthesis was a structural modification of ionic liquids through changing the polarity of imidazolium and pycolinium cations and replacing halide anions with well known antifungal anions (cinnamate, caffeate and mandelate). The findings clearly suggest that the type of alkyl chain on the cation is the most determining factor for IL toxicity. In order to examine how IL structure affects their toxicity towards Fusarium genus, lipophilic descriptor AlogP is calculated from density functional theory and correlated with Fusarium growth rate. All these results demonstrate the high level of the interdependency of lipophilicity and toxicity for investigated ILs towards the Fusarium genus. The data collected in this research suggest that the inhibitory influence of ILs is more pronounced in the case of F. oxysporum.

Full text of DOI:10.1039/c9ra02521a

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Correlation between lipophilicity of newly
synthesized ionic liquids and selected Fusarium
genus growth rate†
Cite this: RSC Adv., 2019, 9, 19189
a
a
b
a
Milan Vranes,
Aleksandar Tot, * Jasenka C´ osic, Snezana Papovic,
´ ˇ ´
ˇ
a
a
c
b
Jovana Panic, Slobodan Gadzuric,
Nenad Jankovic
and Karolina Vrandecic
´
ˇ
´
´
ˇ ´
The purpose of the present study was to examine the effectiveness of 23 different synthesized ionic liquids
ILs) on Fusarium culmorum and Fusarium oxysporum growth rate. The strategy of IL synthesis was
(
a structural modification of ionic liquids through changing the polarity of imidazolium and pycolinium
cations and replacing halide anions with well known antifungal anions (cinnamate, caffeate and
mandelate). The findings clearly suggest that the type of alkyl chain on the cation is the most
determining factor for IL toxicity. In order to examine how IL structure affects their toxicity towards
Fusarium genus, lipophilic descriptor A log P is calculated from density functional theory and correlated
with Fusarium growth rate. All these results demonstrate the high level of the interdependency of
lipophilicity and toxicity for investigated ILs towards the Fusarium genus. The data collected in this
research suggest that the inhibitory influence of ILs is more pronounced in the case of F. oxysporum.
Received 3rd April 2019
Accepted 11th June 2019
DOI: 10.1039/c9ra02521a
rsc.li/rsc-advances
2,5,6
wheat, corn, rice and arable crops.
Integrated plant protec-
1
. Introduction
tion, which includes agronomic techniques, chemical control
Fusarium genera contain over 1000 species, some of which are and the growth of resistant genotypes, is probably the most
recognized as serious pathogens that colonize plants and effective approach to control Fusarium diseases. To avoid the
1,2
produce various mycotoxins. These lamentous fungi are possible development of fungal resistance and preserve the
widespread and cause a wide range of diseases with devastating natural ecosystems and the health of consumers, we need to
3
7,8
yield quantity and quality losses. Fusarium culmorum (W. G. reduce the use of synthetic fungicides as much as possible.
Smith) Sacc. and Fusarium oxysporum Schl. are among the most
Within the last decade, ionic liquids (ILs, currently dened
destructive Fusarium species and play an essential role in the as salts melting below 100 C) have been studied for a various
ꢀ
9,10
etiology of a wide range of cereal, arable crop, fruit, ornamental number of applications where reduced toxicity is demanded.
4
and vegetable diseases. F. culmorum is a soil-borne pathogen The main reason to favor ionic liquids as green solvents was low
and one of the most important corn and small-grain cereal vapor pressure leading to insignicant environmental impact
11–13
pathogens in the cooler temperate climates of Europe, Aus- on air.
However, with rise of scientic interest in ILs, their
tralia, West Asia, and North Africa. In addition, these species environmental friendly potential become more and more under
14–19
also infect numerous other monocotyledonous and dicotyle- question.
Nowadays, the established opinion about their
donous plant species such as potato, watermelon, carnation, “greenness” is that IL properties must be assessed on a case-by-
gladiolus, etc. F. oxysporum is soil-borne species complex case basis. According to this concept, the most promising
distributed all over the world which consists of pathogenic and strategy is the use of renewable resources for the synthesis of
nonpathogenic strains. Pathogenic strains infect almost 150 ILs. The potential of incorporation the well-known biological
plant species including the most important cultivated crops: active ions in ionic liquids can lead to new possible applica-
tions. For example, the antimicrobial properties inherent to
some ILs have been recognized as valuable tools for biocide
a
Faculty of Sciences, Department of Chemistry, Biochemistry and Enviromental
applications, including the development of bioactive coatings
Protection, University of Novi Sad, Trg Dositeja Obradovi ´c a 3, 21000, Novi Sad,
Serbia. E-mail: aleksandar.tot@dh.uns.ac.rs; Fax: +381 21 454 065; Tel: +381 21 against different pathogens, antiseptics, antifouling or anti-
20–23
4
852744
cancer agents.
The promising eld that can have operational
b
Faculty of Agrobiotechnical Sciences in Osijek, University of Josip Juraj Strossmayer in
Osijek, Vladimira Preloga 1, 31000 Osijek, Croatia
benets from a combination of biological activity and IL is
24
agrochemistry. For instance, a hydrophobic IL form of an
c
Department of Chemistry, University of Kragujevac, Faculty of Science, Radoja
agrochemical could improve rain-fastness, reduce dri and the
number of chemicals which need to be applied. Increased
curative activity against plant pathogens might result from re-
Domanovi ´c a 12, 34000 Kragujevac, Serbia
†
Electronic supplementary information (ESI) available: MS, NMR and IR spectra
of the newly synthesized ionic liquids. See DOI: 10.1039/c9ra02521a
This journal is © The Royal Society of Chemistry 2019
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distribution into plant tissue since, in the absence of trans- exchange reaction was performed until a negative reaction on
laminar or systemic movement, a fungicide can only act on the halide ions was achieved using spot test with AgNO3.
target pathogen before it penetrates plant cell.
In the nal stage of synthesis, imidazolium or pycolinium
Additionally, the combination of active cation and anion in hydroxides were slowly titrated by addition of aqueous solutions
one IL formulation might lead to synergistic or antagonistic of mandelic, caffeic or cinnamic acid until inection point was
effects (as observed for pharmaceutically active ILs) which reached. Aer reaching the equimolar ratio of starting
1
5,25,26
might even be used to overcome resistance.
The strategy to compounds, water was removed using vacuum under mild
ꢀ
optimize and control the antifungal activity of ILs is incorpo- conditions (temperature was less than 50 C). The synthesized
ration in their structure compounds that are already commer- ionic liquids were stored under vacuum with P
cially proven in the treatment of fungi.
2
O
5
for the next
With this purpose, 72 h. Water content was determined by the Karl-Fischer coulo-
short chain u-phenylalcanoic/phenylalcenoic acids (mandelic, metric titration and was found to be less than 240 ppm.
27–29
cinnamic and caffeic) were used as an anionic component in
Obtained ILs (structures presented in Fig. 1 and Table S2†)
this work.
were analyzed by MS, NMR and IR spectroscopy to determine
However, the complete mechanism of antifungal action for their structure and purity, and the assignations are presented in
ILs remains unknown, although the most promising quantita- ESI Fig. S1–S63.† The assignation for [OHC mim][Cl] and
NMR spectra were recor-
3
3
0,31
tive descriptor of antimicrobial activity currently indicates the [bmim][Cl] are given in literature.
ꢀ
surface activity. The molecular simulation studies of ionic ded in D
2
O at 25 C on a Bruker Advance III 400 MHz spec-
liquid–biomembrane interactions show the spontaneous trometer. Tetramethylsilane was used as an accepted internal
1
13
1
insertion of cations into the lipid bilayer. The insertion takes standard for calibrating chemical shi for H and C.
H
place regardless of the substituent (alkyl chain length and homodecoupling and 2D COSY method were used routinely for
1
3
polarity), and it changes the structural and dynamic properties the assignation of the obtained NMR spectra. C spectra were
of the bilayer, which is the cause of its disruption. With a goal to assigned by selective decoupling technique. Mass spectrometry
examine the toxic effect of different ILs constituents, as well as was performed by Waters Micromass Quattro II triple quadru-
to nd a correlation between toxicity and structural properties, pole mass spectrometer and MassLynx soware for control and
in this work, the antifungal activities of 23 synthesized ionic data processing. Electrospray ionization in the positive mode
liquids were tested against Fusarium culmorum and Fusarium was used. The electro spray capillary was set at 3.0 kV and the
ꢀ
oxysporum.
cone at 20 V. The ion source temperature was set at 120 C and
ꢁ
1
the ow rates for nitrogen bath and spray were 500 l h and 50 l
ꢁ1
h
, respectively. The collision energy was 20 eV.
2. Materials and methods
2.1. Synthesis
The provenance and purity of reagents used for the synthesis of
ionic liquids are given in Table S1 in ESI† of this manuscript.
The preparation of ILs with the desired anion was performed
using a three-step strategy.
In the rst step, ionic liquids with halide anions were
synthesized and used as starting compounds for further
synthesis. The procedure was based on the reaction of SN2
substitution of adequate heterocycle with appropriate alkyl-
halide or halogenated alcohols. The synthesis of 1-(3-
hydroxypropyl)-3-methylimidazolium chloride was described in
30
our previous paper, while 1-butyl-3-methylimidazolium chlo-
ride was purchased. For the synthesis of pycoline based ionic
liquids, 0.1 mol of methyl-n-pycoline and 0.11 mol of halogen-
oalkane (1-bromobutane or 3-chloro-1-propanol) was dissolved
in toluene in the round bottom ask. The reaction was con-
ꢀ
ducted under atmospheric pressure at a temperature of 125 C
for 72 h. Aer the completion of reaction, toluene was removed
by vacuum. Ionic liquid was washed with ethyl-acetate to
remove potential unreacted starting compounds. Aerward, the
remained solvents were removed using a vacuum and ILs were
2 5
stored under P O for next 48 h.
In the next step, the chloride and bromide ionic liquids were
transferred into hydroxides using ion resin Amberlite IRN 78.
The aqueous solution of ionic liquids was mixed with an
appropriate amount of ion resin under stirring. The ion Fig. 1 Structure and abbreviations of investigated ionic liquids.
19190 | RSC Adv., 2019, 9, 19189–19196
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Table 1 The diameter of fungal growth of F. culmorum and F. oxysporum after treatment with ionic liquids
Diameter of fungal growth (ꢂSD)
IL
48 h
72 h
96 h
120 h
144 h
168 h
192 h
240 h
F. culmorum
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
C
C
C
4
4
4
-2mpyc][Br]
-3mpyc][Br]
-4mpyc][Br]
35.8 ꢂ 3.9
32.8 ꢂ 0.5
32.2 ꢂ 1.7
38.2 ꢂ 1.0
37.2 ꢂ 2.6
38.3 ꢂ 4.4
28.2 ꢂ 2.5
25.5 ꢂ 2.1
23.5 ꢂ 1.3
32.3 ꢂ 3.8
39.0 ꢂ 2.7
35.0 ꢂ 1.0
40.0 ꢂ 3.6
36.8 ꢂ 1.0
37.5 ꢂ 4.1
18.5 ꢂ 1.0
8.5 ꢂ 2.1
65.0 ꢂ 4.8
56.7 ꢂ 1.5
55.2 ꢂ 2.2
68.5 ꢂ 3.1
66.5 ꢂ 3.4
69.0 ꢂ 5.6
51.0 ꢂ 4.7
47.5 ꢂ 2.4
43.8 ꢂ 2.9
61.8 ꢂ 4.2
67.5 ꢂ 3.7
66.0 ꢂ 1.4
68.8 ꢂ 3.8
67.0 ꢂ 1.4
65.0 ꢂ 5.8
32.8 ꢂ 2.6
17.3 ꢂ 3.5
52.5 ꢂ 2.5
58.2 ꢂ 5.9
46.5 ꢂ 3.9
25.0 ꢂ 3.5
78.0 ꢂ 2.0
76.5 ꢂ 3.7
56.5 ꢂ 5.3
86.2 ꢂ 3.3
75.5 ꢂ 3.1
73.5 ꢂ 1.7
88.0 ꢂ 1.4
87.8 ꢂ 3.3
86.5 ꢂ 2.6
69.5 ꢂ 6.5
67.0 ꢂ 1.6
64.2 ꢂ 2.2
84.0 ꢂ 4.1
87.5 ꢂ 2.1
89.0 ꢂ 1.5
89.5 ꢂ 1.0
88.8 ꢂ 1.5
86.5 ꢂ 5.7
53.5 ꢂ 1.7
26.0 ꢂ 3.6
72.5 ꢂ 1.7
76.0 ꢂ 4.9
71.0 ꢂ 1.8
39.0 ꢂ 4.1
90.0 ꢂ 0.0
90.0 ꢂ 0.0
83.5 ꢂ 6.2
90.0 ꢂ 0.0
90.0 ꢂ 0.0
90.0 ꢂ 0.0
90.0 ꢂ 0.0
90.0 ꢂ 0.0
90.0 ꢂ 0.0
88.0 ꢂ 4.0
84.7 ꢂ 1.3
85.2 ꢂ 1.3
90.0 ꢂ 0.0
90.0 ꢂ 0.0
90.0 ꢂ 0.0
90.0 ꢂ 0.0
90.0 ꢂ 0.0
90.0 ꢂ 0.0
75.5 ꢂ 1.7
39.0 ꢂ 3.0
90.0 ꢂ 0.0
88.0 ꢂ 4.0
90.0 ꢂ 0.0
55.2 ꢂ 5.2
90.0 ꢂ 0.0
90.0 ꢂ 0.0
90.0 ꢂ 0.0
OHC
OHC
OHC
OHC
OHC
OHC
OHC
OHC
OHC
OHC
OHC
OHC
3
3
3
3
3
3
3
3
3
3
3
3
-2mpyc][Cl]
-3mpyc][Cl]
-4mpyc][Cl]
-2mpyc][Cin]
-3mpyc][Cin]
-4mpyc][Cin]
-2mpyc][Man]
-3mpyc][Man]
-4mpyc][Man]
-2mpyc][Caf]
-3mpyc][Caf]
-4mpyc][Caf]
bmim][Cl]
90.0 ꢂ 0.0
54.7 ꢂ 4.5
bmim][Cin]
bmim][Man]
bmim][Caf]
OHC
OHC
OHC
OHC
71.7 ꢂ 3.5
82.8 ꢂ 2.6
83.0 ꢂ 4.6
90.0 ꢂ 0.0
29.0 ꢂ 2.5
32.8 ꢂ 5.5
25.7 ꢂ 1.9
12.2 ꢂ 5.0
41.0 ꢂ 3.2
47.3 ꢂ 2.2
28.3 ꢂ 2.5
3
3
3
3
mim][Cl]
mim][Cin]
mim][Man]
mim][Caf]
73.5 ꢂ 5.2
Control
F. oxysporum
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
C
C
C
4
4
4
-2mpyc][Br]
-3mpyc][Br]
-4mpyc][Br]
14.5 ꢂ 1.0
15.0 ꢂ 1.4
15.5 ꢂ 1.0
13.5 ꢂ 1.7
12.5 ꢂ 0.6
13.0 ꢂ 1.4
12.8 ꢂ 0.9
10.7 ꢂ 1.3
13.2 ꢂ 0.5
12.0 ꢂ 1.4
12.2 ꢂ 0.5
12.3 ꢂ 0.9
10.8 ꢂ 1.7
13.7 ꢂ 1.9
13.2 ꢂ 1.7
12.5 ꢂ 0.6
9.2 ꢂ 1.0
30.0 ꢂ 1.6
30.7 ꢂ 2.1
30.5 ꢂ 1.3
33.7 ꢂ 3.4
37.3 ꢂ 5.1
39.0 ꢂ 5.8
28.7 ꢂ 1.7
26.2 ꢂ 1.2
25.5 ꢂ 1.3
29.0 ꢂ 0.8
29.0 ꢂ 3.8
26.8 ꢂ 3.1
22.7 ꢂ 2.8
30.8 ꢂ 2.9
29.3 ꢂ 2.2
20.0 ꢂ 0.8
14.7 ꢂ 1.3
23.0 ꢂ 2.2
17.5 ꢂ 3.7
23.0 ꢂ 0.8
15.0 ꢂ 2.2
22.7 ꢂ 2.1
18.2 ꢂ 6.2
41.8 ꢂ 3.9
40.0 ꢂ 2.2
42.3 ꢂ 3.4
42.8 ꢂ 5.7
42.0 ꢂ 5.0
47.8 ꢂ 8.6
50.8 ꢂ 7.1
35.0 ꢂ 5.7
32.8 ꢂ 1.7
33.5 ꢂ 2.6
36.2 ꢂ 4.2
34.8 ꢂ 5.2
32.5 ꢂ 2.5
28.5 ꢂ 5.1
40.2 ꢂ 3.6
35.0 ꢂ 5.7
28.2 ꢂ 2.4
19.8 ꢂ 0.5
28.7 ꢂ 2.9
28.2 ꢂ 3.0
33.7 ꢂ 1.7
21.5 ꢂ 0.6
32.2 ꢂ 3.6
25.0 ꢂ 5.8
53.0 ꢂ 5.3
55.5 ꢂ 3.1
53.7 ꢂ 3.0
56.8 ꢂ 7.9
56.2 ꢂ 8.3
57.7 ꢂ 7.6
62.0 ꢂ 7.5
44.7 ꢂ 7.4
49.2 ꢂ 2.2
41.8 ꢂ 6.5
48.3 ꢂ 4.6
45.2 ꢂ 8.9
44.0 ꢂ 6.3
38.2 ꢂ 8.7
51.7 ꢂ 5.8
46.8 ꢂ 6.2
38.0 ꢂ 2.6
24.5 ꢂ 0.6
37.7 ꢂ 2.0
36.7 ꢂ 2.4
43.2 ꢂ 2.4
27.2 ꢂ 0.9
40.2 ꢂ 3.2
35.0 ꢂ 4.1
60.5 ꢂ 3.3
69.0 ꢂ 2.0
59.0 ꢂ 2.6
61.8 ꢂ 7.9
67.7 ꢂ 10.7
68.8 ꢂ 8.4
71.8 ꢂ 6.7
50.2 ꢂ 10.1
57.2 ꢂ 1.9
49.0 ꢂ 10.1
55.0 ꢂ 6.9
53.0 ꢂ 11.6
52.0 ꢂ 8.1
45.0 ꢂ 11.9
61.2 ꢂ 7.9
55.3 ꢂ 8.2
45.0 ꢂ 4.1
30.7 ꢂ 1.0
48.0 ꢂ 2.8
45.2 ꢂ 2.9
54.5 ꢂ 2.4
32.2 ꢂ 3.3
50.7 ꢂ 2.5
46.5 ꢂ 4.5
73.0 ꢂ 3.6
76.2 ꢂ 2.6
69.7 ꢂ 4.5
72.5 ꢂ 8.8
77.0 ꢂ 9.9
75.5 ꢂ 5.9
76.8 ꢂ 5.0
55.0 ꢂ 12.9
66.5 ꢂ 3.5
56.2 ꢂ 10.4
63.0 ꢂ 7.3
59.5 ꢂ 12.7
59.8 ꢂ 10.6
52.0 ꢂ 14.5
68.3 ꢂ 8.3
63.8 ꢂ 10.3
54.7 ꢂ 0.5
37.7 ꢂ 1.7
57.7 ꢂ 2.1
56.7 ꢂ 3.2
66.2 ꢂ 0.9
38.2 ꢂ 4.9
62.0 ꢂ 1.6
54.7 ꢂ 7.7
84.2 ꢂ 3.3
83.5 ꢂ 5.0
75.0 ꢂ 5.0
78.5 ꢂ 5.8
81.3 ꢂ 7.8
80.2 ꢂ 4.5
82.2 ꢂ 5.9
59.2 ꢂ 13.0
72.0 ꢂ 6.2
64.7 ꢂ 13.4
70.8 ꢂ 7.6
66.0 ꢂ 15.1
67.5 ꢂ 12.5
57.7 ꢂ 16.7
73.5 ꢂ 6.2
71.8 ꢂ 9.0
67.0 ꢂ 3.2
41.7 ꢂ 3.1
64.7 ꢂ 2.1
63.7 ꢂ 3.3
75.5 ꢂ 1.3
43.5 ꢂ 5.8
70.7 ꢂ 1.9
62.7 ꢂ 8.5
88.0 ꢂ 2.4
87.5 ꢂ 3.8
75.0 ꢂ 7.7
78.5 ꢂ 6.1
88.2 ꢂ 3.5
90.0 ꢂ 0.0
90.0 ꢂ 0.0
68.3 ꢂ 14.7
81.5 ꢂ 10.1
75.2 ꢂ 18.1
82.0 ꢂ 5.7
76.2 ꢂ 16.7
80.3 ꢂ 14.7
67.7 ꢂ 19.0
86.2 ꢂ 7.5
79.8 ꢂ 11.8
75.2 ꢂ 4.0
47.0 ꢂ 3.2
71.2 ꢂ 1.7
71.5 ꢂ 1.0
81.0 ꢂ 1.4
48.5 ꢂ 8.1
78.0 ꢂ 3.6
70.5 ꢂ 7.9
90.0 ꢂ 0.0
OHC
OHC
OHC
OHC
OHC
OHC
OHC
OHC
OHC
OHC
OHC
OHC
3
3
3
3
3
3
3
3
3
3
3
3
-2mpyc][Cl]
-3mpyc][Cl]
-4mpyc][Cl]
-2mpyc][Cin]
-3mpyc][Cin]
-4mpyc][Cin]
-2mpyc][Man]
-3mpyc][Man]
-4mpyc][Man]
-2mpyc][Caf]
-3mpyc][Caf]
-4mpyc][Caf]
bmim][Cl]
bmim][Cin]
bmim][Man]
bmim][Caf]
OHC
OHC
OHC
OHC
13.2 ꢂ 1.2
11.2 ꢂ 1.0
13.7 ꢂ 0.5
9.5 ꢂ 1.3
3
3
3
3
mim][Cl]
mim][Cin]
mim][Man]
mim][Caf]
13.2 ꢂ 0.9
10.2 ꢂ 1.5
17.0 ꢂ 1.2
Control
2
.2. Antifungal activity assay
and one type of selected ionic liquid. All tests were performed at
ꢁ
1
a ILs concentration of mg L . Petri dishes were lled with
0 ml of prepared mixture. Agar plugs (4 mm in diameter) of 1
The in vitro antifungal activities of the 23 ionic liquids against F.
oxysporum and F. culmorum (culture collections of Chair of
Phytopathology, Faculty of Agrobiotechnical Sciences Osijek,
Croatia) were conducted. To test growth inhibition of investi-
gated fungi, the Petri dishes (9 cm in diameter) were lled with
a mixture of recently prepared PDA (Difco, Detroit, Michigan)
1
week old fungal cultures were collected with a sterilized needle
and then placed in the center of the Petri dishes. Petri dishes
ꢀ
were kept in a growth chamber at 22 ꢂ 1 C, with 12 h light/12 h
dark regime. Each measurement consists of four replicates. The
radial growth of the fungal colonies was measured 48, 72, 96,
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developed by the standard Boys–Bernardi counterpoise method.
The free energy of solvation was calculated using the continuum
33
solvation model based on density (SMD). According to this
model, the solvation free energy change at 298.15 K can be
dened as the difference between the solvent and gas electronic
energies, necessitating the corresponding gas phase calcula-
34
tion. The geometry optimizations were carried out in the gas
phase, also using water and octanol as solvents, with SMD
model for the overall solvation effect. Based on these results, the
A log P was calculated as:
DGsolðwaterÞ ꢁ DGsolðoctanolÞ
A log P ¼
2
:303RT
where DGsol is the standard-state solvation free energy change of
a given ionic liquid in octanol or water at 298.15 K. The
standard-state solvation free energy is dened as the free energy
of transfer from the gas phase to the condensed phase, under
the standard state conditions.
3
. Results and discussion
Described tests on F. culmorum and F. oxysporum were per-
formed in order to investigate the inuence of the cation
structural modication, as well as the substitution of halide ion
with anion based on already known antifungal compounds, on
the overall ILs antifungal activity and a possibility to ne control
growth of Fusarium. In Table 1 the results of radial growth of
fungal colonies is presented.
Fig. 2 The correlation between lipophilic descriptor (A log P) and
growth rate for all investigated ionic liquids on: (a) F. culmorum and (b)
+
+
3 3
F. oxysporum. (2P ¼ [OHC -2mpyc] ; 3P ¼ [OHC -3mpyc] ; 4P ¼
+
[
OHC
3
-4mpyc] ).
3.1. Inuence of different anion
From Table 1, it can be seen that the pycolinium ionic liquids
with chloride anion shown signicantly lower toxicity
comparing to ILs with the same cation but with anion based on
short chain u-phenylalcanoic/phenylalcenoic acid. In the liter-
ature, using of different ILs containing halide anions do not
shown remarkable toxicity compared to other anions. This is
1
20, 144, 168, 192 and 240 h aer inoculation. PDA without
ionic liquids served as control.
2.3. Statistical analysis
35
also in agreement with the earlier observation by Garcia et al.
All experimental data were presented as diameter of fungal
growth, as the average value of 4 measurements. Experimental
data were analyzed using Origin 8.1 statistical soware. The
statistical analysis of results were performed using one-way
ANOVA (analysis of variance) technique. The signicance of
differences between means was assessed using the Least
Signicant Difference (LSD) test at the 0.05 probability level.
36
and Zhao et al., who showed that the nature of the inorganic
anion has a negligible effect on the ILs toxicity.
+
In the case of imidazolium-based cations, ([bmim] and
+
[OHC
3
mim] ), the caffeate and mandelate anions express
similar toxic effect as chloride. On the other side, in both
investigated ionic liquids based on imidazolium cations, the
most prominent toxic effect express cinnamate. It is interesting
to note the various effects on different Fusarium species. The
addition of caffeate shows the stimulatory effect on F. culmorum
2.4. Computational simulations
The Jaguar 9.0 program (Schr ¨o dinger Materials Science Suite growth at lower exposure time in contrast to the inhibitory effect
015-4 package) was used to perform density functional theory on F. oxysporum. This effect is the most pronounced in the case
2
+
3
(DFT) calculations. The validity of optimized structures was of [OHC mim] cation. The aforementioned hormetic effect
checked by the Hessian analysis, which showed no presence of (i.e., stimulatory effects occurring in response to low levels of
imaginary frequencies. The non-covalent interactions (NCI) in exposure to agents harmful at high levels of exposure) depends
the obtained optimized structures were investigated and visu- both on the nature of the organism and the duration of expo-
3
2
37
alized by the method of Johnson et al. The posterior corrected sure. Similar phenomena were also observed by Ranke et al.
38
hybrid B3LYP-D3 functional was used with basis set 6-31G+ and Stepnowski et al. in their studies on the toxicity of ILs
d,p). The basis set superposition error (BSSE) was used, towards IPC-81 leukemia cells and HeLa cells, respectively.
(
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Fig. 3 The correlation between lipophilic descriptor (A log P) and growth rate of F. culmorum for ionic liquids containing: (a) cinnamate, (b)
mandelate, (c) caffeate and (d) chloride anion.
3
.2. Inuence of different cation
butyl group leads to destabilization of the lipid double layer
membrane, which can be signicantly reduced through the
introduction of the oxygen as a hydroxyl group in the alkyl side
chain. Furthermore, from obtained results, it is suggested that
the type of alkyl chain on cation ring is the most determining
factor for ILs toxicity. It is obvious that imidazolium ring is far
more harmful towards Fusarium species than pycolinium ring.
These results are in good accordance with literature suggesting
As can be seen from Table 1, for all examined ionic liquids the
most prominent toxic effect was expressed in the case of
+
[bmim] cation, as it was expected. The substitution on the
terminal methyl group of the alkyl chain of the imidazolium
ring with high polar –OH group signicantly reduces the
harmful effect of ionic liquids. Further, by changing the imi-
dazolium to the pycolinium cation, inhibition of Fusarium
growth was reduced even more. The same trend of less harmful
ionic liquids with the introduction of the hydroxyl group in alkyl
side chain can also be observed in the case of pycolinium ionic
liquids. The ne-tuning of pycolinium structure, through
methyl group position changing, shown an incoherent pattern
for toxicity effect. This kind of structural change can be
considered insignicant comparing to changes of alkyl side
chain polarity and type of cation ring.
From the results presented in this work, it is obvious that
alkyl side chain affects the toxicity of both imidazolium and
pycolinium based ionic liquids, due to the change of their
polarity. The lipophilic cations are adsorbed or intercalated in
the cell membrane, causing “perturbations” in the membrane.
This phenomenon can be manifested by expansion or swelling,
increase in uidity, lowering of the phase transition tempera-
ture and alteration of the ion permeability of the membrane.
The presented results indicate that the presence of a lipophilic
28,39,40
the prominent toxic effect of the imidazolium core.
3.3. The correlation between lipophilicity and growth rate
In the literature, the lipophilicity was proposed to determine the
solubility of short-chain u-phenylalcanoic/phenylalcenoic acids
in bacterial membranes and thus inuence their antimicrobial
41,42
activity.
Also, the recent research concerning ionic liquids,
points out lipophilicity as one of the most important factors
39,43,44
deciding their toxicity.
Thus, the establishing of adequate
lipophilic descriptor is of great importance. In order to deduct
more precise conclusion about structural changes of ionic
liquids on toxicity towards Fusarium species, the molecular
descriptor A log P was predicted from DFT calculations.
On the other hand, the radial growth measurement of the
fungal colonies offers only a qualitative description of toxicity.
To have an opportunity to distinguish more precisely inhibition
effect of various ionic liquids on Fusarium growth it is necessary
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Fig. 4 The correlation between lipophilic descriptor (A log P) and growth rate of F. oxysporum for ionic liquids containing: (a) cinnamate, (b)
mandelate, (c) caffeate and (d) chloride anion.
To validate the correlation between two descriptors, growth
rate and A log P, the results for all investigated ionic liquids are
presented in Fig. 2. The Person's regression coefficient obtained
using linear t for F. culmorum is 0.859 and for F. oxysporum
0.836.
From Fig. 2 it can be seen a trend of increasing growth rate
with lower values of A log P. Namely, a higher value of growth
rate indicating the less inhibitory inuence of an investigated
ionic liquid, i.e., lower toxicity. On the other hand, the more
positive values of A log P describes more lipophilic nature of the
compound. All these results demonstrate the high level of the
interdependency of lipophilicity and toxicity for investigated
ionic liquids towards the Fusarium genus.
Fig. 5 The optimized structures of ionic liquids along with represen-
tation of non-covalent interactions: (a) [OHC
OHC -3mpyc][Caff] and (c) [OHC -4mpyc][Caff].
From Fig. 2 it can be seen that ionic liquids with cinnamate
3
-2mpyc][Caff], (b) suppress the growth of Fusarium more than other ionic liquids,
due to the highest lipophilicity. On the contrary, in the experi-
ments with the most hydrophilic ionic liquids containing
chloride anions, the growth of fungi was more rapid. The
[
3
3
to quantify experimental results. Therefore, in this work, the
growth rate was calculated as a derivation of radial growth with
observed time. In literature was conrmed that this parameter
could describe a quite accurately quantitative result of disc
comparison between results obtained for ILs with cinnamate
and caffeate indicate that the presence of –OH group in phenyl
ring signicantly promotes the growth of Fusarium due to
increased polarity. Furthermore, the reduction of double bond
45
dilution method.
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strongly affects antibacterial activity, which is manifested by hydrophilic substance, [OHC
3
-mim][Cl], reduce growth rate
less toxicity of mandelates compared to cinnamates.
signicantly more than their A log P values were predicting.
Comparing IL's effect on the growth of F. culmorum and F. Hence, these results are considered as outliers. From this
oxysporum, it can be seen that the inhibitory inuence of ionic observation, the proposed model cannot be applied for accurate
liquids is more pronounced in the case of F. oxysporum. This prediction of ILs toxicity based on A log P value. In conse-
result suggests that even though both fungi belong to the same quence, besides the lipophilicity based toxic interaction with
genus, there are species specicity concerning interactions membranes, the possibility of further and more specic modes
between ILs and cell membranes and their effect on overall of toxic action has to be taken into account in order to obtain
fungi growth. The cell membrane of F. oxysporum is more a more precise model. Therefore, for compounds with high
sensitive to the presence of ionic liquids and consequently more hydrophilicity, it is necessary to develop a new model based on
prone to incorporate lipophilic compounds in its structure the more detailed investigation.
leading to disruption of cell wall and dysfunctionality of cell,
leading to a smaller growth rate.
4
. Conclusions
In order to better distinguish the individual effect of various
cation on the growth of Fusarium, the variation of A log P vs.
growth rate for each anion is presented in Fig. 3 and 4. As it was
The data collected in this research for pycolinium ILs shown
signicantly lower toxicity comparing to imidazolium ILs
towards Fusarium oxysporum and Fusarium culmorum growth
rate. The Fusarium genus growth rate was the less affected by
chloride anion in comparison with well known antifungal
anions, while ILs with cinnamate proved the most toxic to both
types of fungi. Further, the type of alkyl chain on cation ring
exhibited as the most determining factor for ILs toxicity. The
+
already mentioned in all ILs, [bmim] cation express the most
reductive effect towards Fusarium genus, due to highest A log P
value. On the contrary, the fastest growth of investigated fungi
was obtained aer treatment with imidazolium ionic liquids
with hydroxypropyl side chain and the most negative A log P
values.
3
The cations with similar values of predicted polarity [OHC -

ndings clearly suggest that tuning of the lipophilicity of imi-
+
+
3
3
mpyc] and [OHC -4mpyc] shown almost same effect on F.
dazolium and pycolinium cations by the introduction of polar
groups in the side alkyl chain have pronounced effect on
Fusarium growth rate. Also, it can be concluded that the
inhibitory inuence of ILs is more pronounced in the case of F.
oxysporum. It was shown that is established good correlation
between calculated A log P and experimentally obtained Fusa-
rium genus growth rate. A log P calculated in this work could be
a good predicting tool of antifungal activity.
culmorum growth rate, and more divergent on F. oxysporum. The
only exception occurs in the case of ionic liquids [OHC -3mpyc]
3
[
Caff] and [OHC -4mpyc][Caff] which shown different values of
3
A log P. The explanation for this situation can be found in
optimized geometrical structures of both cations with caffeate
(Fig. 5).
From Fig. 5 it can be seen that the methyl-group on position
C4 inuence different orientation of pycolinium cation towards
caffeate anion, leading to divergent interactions than in case of
methyl-group on position C3. Namely, the –COO from caffeate
is more accessible for interaction with [OHC -3mpyc] , leading
ꢁ
Conflicts of interest
+
3
Authors have no any conict of interest to declare.
to stronger interactions between the polar carboxyl group and
cation. This cause the better shielding of –COO a group which
ꢁ
additionally reduce accessible hydrophilic part of the molecule. Acknowledgements
Thus, interactions between the cell membrane and lipophilic
part of [OHC -3mpyc][Caff] is more possible, leading to
3
a smaller growth rate of F. culmorum and F. oxysporum. This
This work was nancially supported by the Provincial Secre-
tariat for Higher Education and Scientic Research of APV
under project contract 142-451-2766/2018-01/02, the Ministry of
Education, Science and Technological Development of the
Republic of Serbia under project contract ON172012.
observation can also be applied to [OHC -2mpyc][Caff] which
3
have the smallest amount of non-covalent interactions between
cation and carbonyl group of caffeate.
3
Consequently, hydrophilic parts of [OHC -2mpyc][Caff] are
most available for interactions, reducing the possibility of
incorporation of this ionic liquid in the cell membrane. As
a rule, the methylation of pycoline ring on C2 position caused
more hydrophilic properties of ionic liquids, i.e., lower A log P
values comparing to other pycoline based analogs. Due to this
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