This document summarizes research characterizing fatty acid derivatives produced by the fungus Lasiodiplodia theobromae. The researchers identified (Z,Z)-9,12-ethyl octadecadienoate (ethyl linoleate) as one of the most abundant fatty acid esters produced by L. theobromae using NMR and GC-MS. Some of these fatty acid esters, including ethyl linoleate, ethyl palmitate, and ethyl stearate, were found to significantly inhibit or enhance tobacco seed germination and seedling growth at different concentrations, indicating they function as plant growth regulators. This provides new insights into the role of fatty acid esters from L. theob

1. Fatty acid esters produced by Lasiodiplodia theobromae function as
growth regulators in tobacco seedlings
Carla C. Uranga a
, Joris Beld b
, Anthony Mrse b
, Ivan Cordova-Guerrero c
,
Michael D. Burkart b
, Rufina Hernandez-Martínez a, *
a
Centro de Investigacion Científica y de Educacion Superior de Ensenada (CICESE), Carretera Ensenada-Tijuana 3918, Zona Playitas, 22860 Ensenada, B.C.,
Mexico
b
University of California, San Diego, Department of Chemistry and Biochemistry, 9500 Gilman Dr., La Jolla, CA 92093-0358, USA
c
Universidad Autonoma de Baja California (UABC), Calzada Universidad 14418 Parque Industrial Internacional Tijuana, Tijuana, B.C. 22390, Mexico
a r t i c l e i n f o
Article history:
Received 17 February 2016
Accepted 23 February 2016
Available online xxx
Keywords:
Trunk disease fungi
NMR
GC-MS
a b s t r a c t
The Botryosphaeriaceae are a family of trunk disease fungi that cause dieback and death of various plant
hosts. This work sought to characterize fatty acid derivatives in a highly virulent member of this family,
Lasiodiplodia theobromae. Nuclear magnetic resonance and gas chromatography-mass spectrometry of an
isolated compound revealed (Z, Z)-9,12-ethyl octadecadienoate, (trivial name ethyl linoleate), as one of
the most abundant fatty acid esters produced by L. theobromae. A variety of naturally produced esters of
fatty acids were identified in Botryosphaeriaceae. In comparison, the production of fatty acid esters in the
soil-borne tomato pathogen Fusarium oxysporum, and the non-phytopathogenic fungus Trichoderma
asperellum was found to be limited. Ethyl linoleate, ethyl hexadecanoate (trivial name ethyl palmitate),
and ethyl octadecanoate, (trivial name ethyl stearate), significantly inhibited tobacco seed germination
and altered seedling leaf growth patterns and morphology at the highest concentration (0.2 mg/mL)
tested, while ethyl linoleate and ethyl stearate significantly enhanced growth at low concentrations, with
both still inducing growth at 98 ng/mL. This work provides new insights into the role of naturally
esterified fatty acids from L. theobromae as plant growth regulators with similar activity to the well-
known plant growth regulator gibberellic acid.
© 2016 Elsevier Inc. All rights reserved.
1. Introduction
The Botryosphaeriaceae are a family of fungi that have been
found to affect several economically important woody plants
around the world and are considered trunk disease fungi. Some of
the symptoms these fungi cause include gummosis, wedge-shaped
necrotic cankers in tree wood, and stunted growth [1]. Presently, in
Vitis vinifera, Lasiodiplodia theobromae (teleomorph Botryosphaeria
rhodina) has been found to be the most virulent [1,2]. However, it
can also be found as an endophyte or latent pathogen [3]. Many
other Botryosphaeriaceae, including Neofusicoccum parvum, have
been isolated from V. vinifera and other plant species [4].
Characterization of the metabolites produced by L. theobromae
is critical for understanding the metabolic pathways involved
during colonization, as well as for the discovery of novel or inter-
esting compounds. Lipases have an important function in patho-
genicity of fungi (triacylglycerol acyl-hydrolases, E.C. 3.1.1.3) [5].
These enzymes are involved in the degradation of cell membranes
and storage lipids, and esterification of these with alcohols [6]. Li-
pases liberate free fatty acids, which are the starting material for
many secondary metabolites such as oxylipins, studied in other
phytopathogenic fungi, [7e9]. Free fatty acids are also a source of
energy and the acetyl CoA necessary for polyketide-type secondary
metabolites produced by Botryosphaeriaceae [10]. The objective of
this work was to characterize compounds produced or bio-
transformed by L. theobromae in natural substrates, and assess their
effects in plants.
Abbreviations: NMR, nuclear magnetic resonance; GC-MS, gas chromatography-
mass spectrometry; FA, fatty acids; FAE, fatty acid esters; FAME, fatty acid methyl
esters; FAEE, fatty acid ethyl esters; LAEE, linoleate ethyl ester; PAEE, palmitate
ethyl ester; SAEE, stearate ethyl ester; OAEE, oleate ethyl ester; PA, free palmitate;
GA, gibberellic acid.
* Corresponding author.
E-mail addresses: curanga@cicese.edu.mx (C.C. Uranga), joris.beld@drexelmed.
edu (J. Beld), amrse@ucsd.edu (A. Mrse), icordova@uabc.edu.mx (I. Cordova-
Guerrero), mburkart@ucsd.edu (M.D. Burkart), ruhernan@cicese.mx
(R. Hernandez-Martínez).
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
http://dx.doi.org/10.1016/j.bbrc.2016.02.104
0006-291X/© 2016 Elsevier Inc. All rights reserved.
Biochemical and Biophysical Research Communications xxx (2016) 1e7
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2. 2. Materials and methods
Two isolates were kindly provided by Dr. Douglas Gubler from
the University of California at Davis, (USA), L. theobromae
UCD256Ma (isolated in Madera County, California, USA) [11]; and
an isolate of N. parvum, (UCD646So, isolated in Sonoma County).
Isolates used belonging to CICESE at Mexico are: L. theobromae
MXL28, Fusarium oxysporum f. sp. lycopersici, and Trichoderma
asperellum isolated from grapevine, tomato and carnation plants,
respectively.
2.1. Induction of secondary metabolism in L. theobromae
Media for induction of metabolism of both L. theobromae iso-
lates consisted of 25 g ground oatmeal powder and 50 mL Vogel's
salts solution, autoclaved twice. Isolates were inoculated with one
1 cm mycelial disc of the fungi grown in potato dextrose agar (PDA).
As a negative control, oatmeal without fungus was used. Three
biological replicates for the negative controls and the experimental
conditions were set. Samples were incubated at room temperature
(RT) in the dark for a total of 60 days. To assess the production of
compounds of interest in different carbon sources, fungal isolates
were incubated in 50 mL Vogel's minimal media supplemented
with 5% glucose, 5% grape seed oil, 5% glucose þ5% grape seed oil, or
5% fructose. These were incubated in triplicate for 20 days at 25 C
in the dark.
2.2. Solvent extraction of fungal incubations
Before extraction, samples were frozen at À80 C then lyophi-
lized for 48 h. A modified Folch extraction [12] was done using a
solvent mixture of 75 mL dichloromethane (DCM), 75 mL methanol
and 0.01% butylated hydroxytoluene (as antioxidant), and extracted
overnight at 4 C. The samples were placed in a separating funnel to
separate and collect each phase. The organic phases (DCM) were
evaporated with a rotovapor (Buchi R-114) at 45 C and the
remaining oils aliquotted to Eppendorf tubes and stored at À20 C
until analysis.
Thin layer chromatography (TLC) on silica gel sheets (Merck)
with a fluorescence indicator was performed on the crude oil ex-
tracts and developed using 5% ethyl acetate (v/v) in hexane with
two sequential chromatographic developments. The chromato-
grams were stained with vanillin/H2SO4. The oil extract was sepa-
rated on silica gel (Unisil) using step elutions consisting of different
concentrations of ethyl acetate (0, 5, and 10% EtOAc) in hexane.
Fractions with the compounds of interest (eluted in 100% hexane)
were evaporated and re-fractionated by preparative chromatog-
raphy with a C-18 column (Phenomenex), using a gradient of 100%
H2O containing 0.1% formic acid to 100% acetonitrile containing
0.1% formic acid. The compound of interest eluted with 100%
acetonitrile/0.1% formic acid, was collected, evaporated and re-
purified using preparative TLC with a solvent system of 1% ethyl
acetate in hexane. Pure compound, monitored by TLC, was used for
mass spectrometry analysis, GC-MS, proton and carbon NMR.
2.3. Nuclear magnetic resonance (NMR) and mass spectrometry
In order to determine the molecular weight and formula of the
purified compound of interest, high resolution mass spectrometry
was performed on an Agilent 6230 ESI-TOF MS. Proton (1
H) and
carbon (13
C) NMR analyses were obtained from a Varian 500 MHz
instrument equipped with an XSens 2-channel NMR cold probe
optimized for direct observation of 13
C. Data was analyzed with the
program ACD/NMR processor Academic Edition [13].
2.4. In vitro FAE production (Fischer-Speier esterification) of oat
and grapeseed oil
Knowing the nature of the compound, a positive control con-
sisted of an in vitro Fischer-Speier esterification [14] of the oat oil
fraction extracted, and the grape seed oil used in the incubations.
Briefly, 2.5 mL of oil was mixed with 1 mL of ethanol or methanol,
to which five drops of H2SO4 were added as a catalyst. The samples
were placed in sealed glass vials and heated to 100 C for 30 min.
Saturated sodium bicarbonate was added to neutralize the acid, and
the phases containing FAE were collected and analyzed via GC-MS.
2.5. Gas chromatography-mass spectrometry of crude extracts
All samples, including the positive controls, were analyzed for
naturally produced fatty acid ethyl esters by GC-MS. A standard
curve was created from octadecadienoate (Z, Z) ethyl ester (LAEE)
standard (Cayman Chemical) from which concentrations of un-
knowns were calculated. The standard was diluted from 625 mg/L to
40 mg/L in hexanes. Ten mL of all unknowns were dissolved in 1 mL
hexanes without esterification and analyzed by analytical GC-MS on
an Agilent 7890A GC system, connected to a 5975C VL MSD quad-
rupole MS (EI), using helium as the carrier gas and a 60 m DB23
column, with a gradient of 110 Ce200 C at 15 C/min followed by
20 min at 200 C and 20 min at 240 C. All compounds were iden-
tified via NIST library searches, and where applicable, co-injection of
standard and comparison with a 37 FAME mix (SigmaeAldrich).
LAEE, OAEE ((Z)-9-oleate ethyl ester), SAEE (stearate ethyl ester) and
PAEE were purchased as purified standards (Cayman Chemical).
2.6. Effects of FAE on tobacco seed germination and hypocotyl
growth
With the aim to test the effect of the isolated compounds in
planta, we chose tobacco (Nicotiana tabacum), a well-studied plant
model [15]. Seeds were surface-sterilized in 50% household bleach
(8.25% NaOCl) for 1 min and rinsed 3 times with sterile water before
use. Approximately 100 ml packed volume of seeds were placed in
Murashige and Skoog salts (with Gamborg vitamins), 0.8% agar, 3%
sucrose and the antifungal Plant Preservative Mix (PPM, Plant Cell
Technology Inc.), containing 200 mg/mL of either LAEE, PA, PAEE,
OAEE and SAEE emulsified in 0.08% kolliphor-188. All experiments,
including negative controls were done in triplicate under natural
lighting conditions. The length of the hypocotyl was measured after
7e10 days post-sowing by pictures taken with a calibrated
Olympus stereo microscope (SZX12) at 7x magnification, using
Image J software [16]. Seedling lengths (N ¼ 30) from cotyledon tip
to root tip were measured for each experimental condition.
Morphology was assessed and documented 45 days post-dosing
and sowing. Concentration dependence was then studied in Mur-
ashige and Skoog (MS)þ3% sucrose using a concentration range of
3.1 mg/mLe 98 ng/mL for SAEE and LAEE, in triplicate, including
negative controls. Finally, a germination experiment was done us-
ing 1 mg/mL of each FAE, including the known plant growth regu-
lator gibberellic acid (GA, “Supergrow” from Consolidated
Chemical) as a positive control, using MS without sucrose to
resemble field conditions. A one-way ANOVA followed by a Tukey-
HSD post-hoc analysis was performed on the data with p
values 0.05 considered significant, using XLSTAT statistical anal-
ysis software. Graphs were generated with Graphpad Prism soft-
ware. Time-lapse video (Lapse It 2.5 pro) during germination was
taken of a negative control and N. tabacum exposed to 98 ng/mL
SAEE under continuous white light.
Supplementary video related to this article can be found at
http://dx.doi.org/10.1016/j.bbrc.2016.02.104.
C.C. Uranga et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e72
Please cite this article in press as: C.C. Uranga, et al., Fatty acid esters produced by Lasiodiplodia theobromae function as growth regulators in
tobacco seedlings, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.02.104

3. 3. Results
3.1. Metabolite identification
Thin layer chromatography (TLC) of the oil portion from
extraction of L. theobromae incubated in oatmeal revealed a
prominent band that stained dark green/blue with vanillin/H2SO4.
This compound was present in all three biological replicates incu-
bated with L. theobromae and absent in the negative controls
(Fig. 1A). High resolution mass spectrometry of the compound
detected an M þ H ion at 309.278 m/z, the most probable molecular
formula being C20H36O2 (Dataset A in Ref. [17]). Proton and carbon
nuclear magnetic resonance (NMR) spectra (Fig. 1 in Ref. [17])
identified this compound as LAEE. The relative chemical shifts
agree with those published in the literature for LAEE [18,19]. The (Z,
Z)-configuration is evident by the average coupling constant of
multiplet 5.27e5.46 (6.1 Hz), and confirmed to be within the range
for cis hydrogen coupling in double bonds [20,21]. From L. theo-
bromae incubated in 5% glucose þ5% grape seed oil, TLC results
revealed a compound with the same Rf as LAEE. This was confirmed
using GC-MS by co-injection of LAEE standard with the crude
samples. Further GC-MS analysis demonstrated the presence of a
variety of ethyl esters (Table 1) in both isolates of L. theobromae
incubated in oatmeal, not detected in the negative controls. Chro-
matograms may be found in Fig. 2,3,4,5 in Ref. [17].
A four-point standard curve using (Z, Z)-9, 12-octadecadienoate
ethyl ester standard with an R2
value of 1.00 was obtained with the
linear equation y ¼ 3.53E þ 08x À 1.43Eþ06, from which the un-
knowns were calculated. Using oat as substrate, LAEE was the major
FAE produced by L. theobromae. The incubation of L. theobromae for
60 days yielded 20.1 ± 1.3 g/L in UCD256Ma and 28.7 ± 7.1 g/L in
MXL28 for LAEE. LAEE was also detected in strain UCD256Ma
incubated in 5% glucose þ5% grape seed oil, producing 2.4 ± 0.7 g/L
(Fig. 2), as well as in other isolates (Table 1) after 20 days.
No FAE were produced by L. theobromae UCD256Ma when
incubated in 5% fructose. Ethyl palmitate (PAEE) and 1H-2-
Benzopyran-1-one, 3, 4-dihydro-8 hydroxy-3-methyl- (mellein)
were detected in 5% glucose as the sole carbon source (Table 1, Fig. 3
in Ref. [17]) indicating de novo production of PAEE by the fungus.
FAE identified by GC-MS, 37 FAME standard comparison and the
NIST library are listed in Table 1, several of which have not been
reported previously to be produced by these fungi. In grapeseed oil
only, metabolism shifted towards the production of 9-octadece-
noate methyl ester (OAME) (44%) and LAEE (40.9%) (Table 1 in
Ref. [17]) indicating ethanol production in the absence of glucose.
SAEE and PAEE were also produced in the absence of glucose.
3.2. Tobacco seed germination and growth in FAE
Statistically significant inhibitory effects on seed germination
were observed by all FAE compounds tested at 0.2 mg/mL as
compared to the negative controls except for free palmitate (PA)
and PAEE in MS without sucrose (Fig. 2A and B). In MS without
sucrose, LAEE, OAEE, SAEE and the crude oil caused seedling growth
inhibition. In MSþ3% sucrose, LAEE, PAEE, OAEE, SAEE and the
crude extract inhibited growth. However, both LAEE and SAEE
induced growth at lower concentrations in MSþ3% sucrose (Fig. 2C
and D). The effect was clear for LAEE, which increased seedling
length at 98 ng/mL. SAEE induced growth at the lowest concen-
trations tested, with more variability between concentrations.
Time-lapse video shows a faster germination rate in N. tabacum
exposed to 98 ng/mL SAEE under continuous white light (Video file
1), indicating light to be a factor in this process.
Leaf morphology of tobacco seedlings germinated in FAE in
Fig. 1. Identification of the compound isolated and characterized from L. theobromae. A: TLC of the negative control (C-) and the compound of interest (Cþ, black arrow). B: GC-MS of
compound isolated from L. theobromae. C: NIST library standard match of the unknown to LAEE. D: Graph of LAEE quantification from incubations in oatmeal or 5% glucoseþ 5%
grapeseed oil, error bars represent standard error of the mean.
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4. MSþ3% sucrose was also affected. Leaves showed abnormal elon-
gation and bifurcation when exposed to the crude extract or
0.2 mg/mL SAEE, and expanded cotyledons with abnormal elon-
gation of the first true leaf at lower SAEE concentration (3.1 mg/mL).
Tobacco seedlings exposed to LAEE, PAEE, SAEE and the crude
extract during germination showed stunted growth and chlorosis
(Fig. 3). More examples of effects may be found in Fig. 8, 9 in
Ref. [17].
Table 1
Fatty acid esters (FAE) from isolates of Lasiodiplodia theobromae, Neofusicoccum parvum, Fusarium oxysporum and Trichoderma asperellum, identified via GC-MS, 37 FAME
standard and NIST library comparison. Average areas under the curve values from triplicates are shown for the incubations with fungal isolates. ChEBI identifications are shown
where applicable.
Compound Positive
control, fischer
esterification
L. theobromae incubation
in oatmeal 60 days
5%
Glucose
5% grape-seed
oil
Incubation in 5% glucoseþ5% grape
seed oil, 20 days
RT
Oat
oil
Grape seed
oil
UCD 256
Ma
MXL28 UCD
256Ma
UCD 256Ma UCD256Ma N. parvum
UCD646So
F. ox T. as
Methyl hexadecanoate ChEBI:69187 6 Â 106
1.4 Â 106
1.4 Â 106
2.6 Â 106
N/D 1.7 Â 105
N/D N/D N/D N/D 12.7
Ethyl hexadecanoate (PAEE)
ChEBI:84932
3.2 Â 107
1.9 Â 106
5.2 Â 107
6.6 Â 107
9.1 Â 104
1 Â 105
2.6 Â 106
2.3 Â 106
7 Â 105
9.2 Â 104
13.3
Hexadecanoate, 2-methylpropyl ester N/D N/D 9.5 Â 104
1.2 Â 106
N/D N/D N/D N/D N/D N/D 15.7
9-Octadecenoate (Z)- methyl ester
ChEBI:27542
9.8 Â 106
2.4 Â 107
1.1 Â 106
1.1 Â 106
N/D 2.8 Â 106
3.1 Â 105
2 Â 106
N/D 6.9 Â 105
17.0
Octadecanoate ethyl ester (SAEE)
ChEBI:84936
1.8 Â 106
7.9 Â 105
1.8 Â 106
2.2 Â 106
N/D 1.3 Â 104
1.6 Â 106
7.9 Â 105
7.7 Â 104
N/D 17.3
9-Octadecenoate (Z), ethyl ester
(OAEE) ChEBI:84940
5.3 Â 107
3.2 Â 107
3.1 Â 107
4.2 Â 107
N/D N/D 3.0 Â 107
2.3 Â 107
2.4 Â 105
2.3 Â 106
18.1
9-Octadecenoate (E) ethyl ester 1.1 Â 106
2.5 Â 105
1.5 Â 106
3.4 Â 106
N/D N/D 3.9 Â 105
3.3 Â 105
N/D N/D 18.3
9,12-Octadecadienoate (Z,Z)-, methyl
ester ChEBI:69080
2.3 Â 107
7 Â 106
2 Â 106
3.1 Â 106
N/D 6 Â 105
2.7 Â 104
5.7 Â 105
N/D N/D 18.4
9,12-Octadecadienoate (Z,Z) ethyl
ester (LAEE) ChEBI:31572
1.1 Â 108
8.9 Â 106
7 Â 107
1 Â 108
N/D 2.6 Â 106
6.9 Â 106
9.1 Â 106
5.5 Â 105
2.3 Â 105
19.6
9,12,15-Octadecatrienoate
(Z,Z,Z)-ethyl ester)
ChEBI:84851
2.0 Â 106
1.7 Â 105
1.3 Â 106
2.2 Â 104
N/D 7.6 Â 104
7.4 Â 105
2.8 Â 105
N/D N/D 21.7
2H-1-Benzopyran, 3,4-dihydro-
(R ± mellein)
N/D N/D N/D 6.9 Â 105
4.5 Â 105
N/D N/D N/D N/D N/D 23.6
N/D: not detected; F.ox: Fusarium oxysporum; T.as: Trichoderma asperellum; RT: Retention time. Negative controls did not yield FAE, therefore are not included in the table. See
Fig. 2A in Ref. [17].
Fig. 2. Concentration dependence of FAE on tobacco seed germination rates. Seedling length 7e8 days post-planting, N ¼ 30 for each condition, using a one-way ANOVA and a post-
hoc Tukey-HSD analysis, p-value 0.05. A; 0.2 mg/mL FAE or crude oil extract in MS only. B; 0.2 mg/mL FAE or crude oil extract in MSþ3% sucrose. C; LAEE 3.1 mg/mLÀ98 ng/mL in
MS-3% sucrose. D; SAEE 3.1 mg/mL- 98 ng/mL in MSþ3% sucrose. Letters above graphs indicate statistically significant differences or similarities between experimental conditions.
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5. In the final germination experiment, all FA and FAE were found
to induce germination at 1 mg/mL to varying degrees (Fig. 4). SAEE
and LAEE induced germination similarly to gibberellic acid.
4. Discussion
Fatty acids and modified fatty acids are important molecules
during colonization of plants by pathogenic fungi, serving diverse
functions such as energy-sources, signaling, and virulence factors
[8]. L. theobromae naturally produces a variety of FAE in plant-
derived triglycerides. This is the first report of their production in
L. theobromae, and the other fungi studied.
Two Botryosphaeriaceae were found to be able to produce a
wider variety and higher quantities of FAE than the rest of the
tested fungi. FAE production in T. asperellum was lower than L.
theobromae and N. parvum. The FAE that affect growth regulation in
tobacco were produced in higher abundance by the trunk disease
Fig. 3. Morphology of N. tabacum germinated with LAEE, SAEE, PAEE or crude extract of L. theobromae 45 days post-sowing and dosing. A; Negative control. B; 0.2 mg/mL crude
extract from L. theobromae incubated in 5% glucoseþ5% grapeseed oil. C; 0.2 mg/mL LAEE: D; 0.2 mg/mL PAEE. E; 0.2 mg/mL SAEE. F; 3.1 mg/mL SAEE.
Fig. 4. N. tabacum exposed to 1 mg/mL FAE during germination in MS without sucrose.
Gibberellic acid (GA) serves as a positive control for comparison. Letters above graphs
indicate statistically significant differences or similarities between experimental
conditions.
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6. fungi, and barely by the other tested fungi. For example, SAEE was
not produced by T. asperellum, and F. oxysporum produced more
PAEE than T. asperellum. These differences in fatty acid metabolism
may be a factor that differentiates trunk disease fungi from other
phytopathogenic fungi, or from non-phytopathogens such as T.
asperellum.
FAE may act in a variety of plant growth processes. FAE are
known to have various functions in eukaryotes, activating steroid
hormone receptors in humans [22], or inducing apoptosis [23]. LAEE
and SAEE have been extracted from plants such as Allium sativum
(garlic) [18], the purple shamrock Oxalis triangularis [19], and the
medicinal plant Moringa oleifera [24]. The biosynthetic machinery
leading to these compounds in the plant may involve pyruvate
decarboxylase, alcohol dehydrogenase and lipases [25,26]. Plants
are able to ferment glucose for energy production during flower and
pollen development [27e29], and oxygen levels at the ovary are
known to be at zero [30]. Hypoxia is part of dormancy and dormancy
release in V. vinifera [25] and other fruiting trees, with the produc-
tion of cyanogenic glucosides and starch breakdown linked to the
onset of flowering [31,32]. Hypoxia is also induced in agriculture in
grapevines and other trees to artificially stimulate bud break and
increase agricultural yields with the use of hydrogen cyanamide
[33]. Another cause of low oxygen levels in the plant is excess
watering of roots [34]. Since L. theobromae has an endophytic phase
in the plant, both natural dormancy periods and chemically induced
hypoxia in V. vinifera or other trees may provide the fungus with the
habitat that promotes anaerobic fermentation, resulting in the
production of ethanol and other alcohols required for fungal lipases
to esterify these to free fatty acids.
Fungi may be using FAE to manipulate plant growth. The ability
of fungi to affect plant growth has been observed in the fungus
Gibberella fujikuroi, which is known to produce gibberellic acid, as
well as in the fungus Botrytis cinerea, which produces abscisic acid
[35,36]. In this work it was shown that Botryosphaeriaceae are able
to produce higher quantities and a wider variety of FAE as
compared to the other fungi studied. LAEE, and SAEE were found to
have significant physiological effects in tobacco, acting as growth
regulators during germination and early growth, on par with gib-
berellic acid at 1 mg/mL. Although much work remains to be done to
understand the detailed physiological routes affected in the plant, it
is proposed that fatty acid esters be considered plant growth reg-
ulators due to their ability to affect tobacco germination and early
growth.
Acknowledgements
Thanks to CONACyT and UCMEXUS, who provided a doctoral
stipend for Carla C. Uranga. Thanks to Dr. Yongxuan Su from the
small molecule mass spectrometry department at UCSD, Eduardo
Morales and Dr. Manuel Segovia from CICESE, special thanks to Dr.
Katrin Quester from the UNAM in Ensenada for instrumentation
support. Thanks to Dr. James Nowick from the University of Cali-
fornia, Irvine for his support with NMR analysis in this work, and
special thanks to Claudio Espinosa de los Monteros for help with
figure graphic design.
Transparency document
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online at http://dx.doi.org/10.1016/j.bbrc.2016.02.104.
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