On the metabolites produced by Colletotrichum gloeosporioides a fungus proposed for the Ambrosia artemisiifolia biocontrol; spectroscopic data and absolute configuration assignment of colletochlorin A

Article abstract of DOI:10.1080/14786419.2017.1385020

Ambrosia artemisiifolia L. is responsible for serious allergies induced on humans. Different approaches for its control were proposed during the COST Action FA1203 “Sustainable management of Ambrosia artemisiifolia in Europe” (SMARTER). Fungal secondary metabolites often show potential herbicidal activity. Three phytotoxins were purified from the fungal culture filtrates of Colletotrichum gloeosporioides, isolated from infected leaves of A. artemisiifolia. They were identified by spectroscopic and chemical methods as colletochlorin A, orcinol and tyrosol (1, 2 and 3). The absolute configuration 6’R to colletochlorin A was assigned for the first time applying the advanced Mosher’s method. When assayed by leaf-puncture on A. artemisiifolia only 1 caused the appearance of large necrosis. The same symptoms were also induced by 1 on ambrosia plantlets associated with plant wilting. On Lemna minor, colletochlorin A caused a clear fronds browning, with a total reduction in chlorophyll content.

Full text of DOI:10.1080/14786419.2017.1385020

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
On the metabolites produced by Colletotrichum
gloeosporioides a fungus proposed for the
Ambrosia artemisiifolia biocontrol; spectroscopic
data and absolute configuration assignment of
colletochlorin A
Marco Masi, Maria Chiara Zonno, Alessio Cimmino, Pierluigi Reveglia,
Alexander Berestetskiy, Angela Boari, Maurizio Vurro & Antonio Evidente
To cite this article: Marco Masi, Maria Chiara Zonno, Alessio Cimmino, Pierluigi Reveglia,
Alexander Berestetskiy, Angela Boari, Maurizio Vurro & Antonio Evidente (2017): On the
metabolites produced by Colletotrichum gloeosporioides a fungus proposed for the Ambrosia
artemisiifolia biocontrol; spectroscopic data and absolute configuration assignment of colletochlorin
A, Natural Product Research, DOI: 10.1080/14786419.2017.1385020
To link to this article: http://dx.doi.org/10.1080/14786419.2017.1385020
Published online: 13 Oct 2017.
Submit your article to this journal
Article views: 10
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=gnpl20
Download by: [Eastern Michigan University]
Date: 16 October 2017, At: 01:30

Natural Product research, 2017
https://doi.org/10.1080/14786419.2017.1385020
On the metabolites produced by Colletotrichum
gloeosporioides a fungus proposed for the Ambrosia
artemisiifolia biocontrol; spectroscopic data and absolute
configuration assignment of colletochlorin A
Marco Masia , Maria Chiara Zonnob, Alessio Cimminoa , Pierluigi Revegliaa,
Alexander Berestetskiyc, Angela Boarib, Maurizio Vurrob and Antonio Evidentea
adepartment of chemical sciences, university of Naples Federico II, complesso universitario Monte s. angelo,
c
Napoli, Italy; bInstitute of sciences of Food Production, National research council, Bari, Italy; all-russian
Institute of Plant Protection, Federal agency of scientific organizations, Pushkin, saint-Petersburg, russia
ABSTRACT
ARTICLE HISTORY
received 19 July 2017
accepted 14 september 2017
Ambrosia artemisiifolia L. is responsible for serious allergies induced
on humans. Different approaches for its control were proposed during
the COST Action FA1203 “Sustainable management of Ambrosia
artemisiifolia in Europe” (SMARTER). Fungal secondary metabolites
often show potential herbicidal activity. Three phytotoxins
were purified from the fungal culture filtrates of Colletotrichum
gloeosporioides, isolated from infected leaves of A. artemisiifolia.
They were identified by spectroscopic and chemical methods
as colletochlorin A, orcinol and tyrosol (1, 2 and 3). The absolute
configuration 6’R to colletochlorin A was assigned for the first time
applying the advanced Mosher’s method. When assayed by leaf-
puncture on A. artemisiifolia only 1 caused the appearance of large
necrosis. The same symptoms were also induced by 1 on ambrosia
plantlets associated with plant wilting. On Lemna minor, colletochlorin
A caused a clear fronds browning, with a total reduction in chlorophyll
content.
KEYWORDS
Ambrosia artemisiifolia;
Colletotrichum
gloeosporioides; biocontrol;
phytotoxins; colletochlorin a,
orcinol and tyrosol
1. Introduction
Ambrosia artemisiifolia L., also known as common ragweed, is a widespread invasive weed
native to North America. This plant is very competitive and forms large and almost pure
populations in natural or uncultivated areas, gardens, railway ballast, yards, roadsides and
CONTACT antonio evidente
evidente@unina.it
© 2017 Informa uK limited, trading as taylor & Francis Group

2
M. MASI ET AL.
cultivated fields after harvest (Essl et al. 2015). The major concern regarding common rag-
weed is its highly allergenic pollen that causes sensitisation of the population generating
huge medical costs. Moreover, ragweed is becoming a major weed causing severe crop
losses and troubles for its control (Gerber et al. 2011).
Chemical herbicides and mechanical control are effective as short-term measures in small
infested areas, but are rather insufficient in larger areas and in natural environments, where
these methods are unable to prevent flowering, pollen dispersal and seed production, or
cannot be used due to environmental issues and safety restrictions. Biological control with
the use of microorganisms or their toxic metabolites has been considered a potential alter-
native approach for managing weeds (Cimmino et al. 2015a) and can be applied to control
the common ragweed. At this regard, a pathogenic fungus responsible for necrotic spots of
A. artemisiifolia was previously isolated from naturally diseased leaves and stems, and iden-
tified as Colletotrichum gloeosporioides (Berestetskiy, unpublished).
Colletotrichum is a fungal genus comprising a large number of endophytic, saprophytic
and plant pathogenic species. It is one of the most economically important plant pathogenic
genera, responsible of anthracnose disease of fruits and leaves in a wide range of hosts, with
conspicuous crop losses (Sutton 1992; Phoulivong et al. 2010). For this reason, species
belonging to this genus have been subjected to extensive studies involving their pathogen-
esis, morphology, multigene analysis, physiology, host range and disease life cycle. Due to
the severe diseases they cause, to the fast symptom appearance, and to their fast growth
and easiness to produce conidia in vitro, species of this genus had been proposed for bio-
logical control of a number of important weeds, such as hemp sesbania (Sesbania exaltata)
with Colletotrichum truncatum (Boyette et al. 1993), or velvetleaf (Abutilon theophrasti) by
Colletotrichum coccodes (Wymore et al. 1988). Two formae speciales of C. gloeosporioides,
namely malvae and cuscutae, where also commercialised in the past as mycoherbicides for
the control of Malva pusilla (BioMal) and Cuscuta sp. (Lubao), respectively, demonstrating
the potential of the genus for weed biocontrol (Committee on Mycoherbicides for Eradicating
Illicit Drug Crops 2011).
Due to the pathogenic characteristics of these fungi, the studies on the production, iden-
tification and chemical and biological characterisation of their secondary metabolites
received great attention. However, due to the size of the genus, counting hundreds of species
and formae speciales, this enormous source of bioactive metabolites has been only partially
explored (García-Paión and Collado 2003). Recently, extensive work carried out on
Colletotrichum higginsianum led to the isolation from the mycelium of two new diterpenoid
α-pyrones, named higginsianins A and B, showing antiproliferative activity against a panel
of six cancer cell lines (Cimmino et al. 2015b). Furthermore, two new tetrasubstituted pyran-
2-one and dihydrobenzofuran, named colletochlorins E and F, which showed phytotoxic
activity on Sonchus arvensis, Lemna minor and Phelipanche ramosa, were isolated from the
culture filtrates of the same fungus (Masi et al. 2017).
In preliminary observations, the ability of a strain of C. gloeosporioides to produce in vitro
phytotoxic metabolites was ascertained (Zonno et al. 2015, 2016).
In the present study, thanks to the bioactivity-guided fractionation of the cultural organic
extract, three metabolites were isolated and identified as colletochlorin A, orcinol and tyrosol.
The manuscript reports their isolation, the evaluation of their phytotoxic activity and dis-
cusses the potential use in weed biological control. Furthermore, the complete NMR

NATURAL PRODUCT RESEARCH
3
spectroscopic characterisation and the absolute configuration assignment of colletochlorin
A is also reported.
2. Results and discussion
The organic extract obtained from C. gloeosporioides culture filtrates was purified as reported
in the Experimental section. Three pure metabolites, namely colletochlorin A, orcinol and
tyrosol (1–3, Figure 1) were isolated and identified by comparison of their physic and spec-
troscopic (1H NMR and MS) data with those previously reported in the literature (Kimura and
Tamura 1973; Kosuge et al. 1973, 1974; Mori and Sato 1982; Capasso et al. 1992; Monde et
al. 1998; Cimmino et al. 2017a).
1
The H NMR spectrum of 1 was very similar to that of both the natural and synthetic
colletochlorin A (Kosuge et al. 1974; Mori and Sato 1982). The chemical shifts of all colle-
tochlorin A carbons were assigned for the first time based on the couplings observed in the
HSQC and HMBC spectra (Berger and Braun 2004) as reported in Table 1. In particular, the
coupling observed in the HSQC spectrum allowed to assign the signals resonating at δ 194.5,
121.6, 79.2, 35.9, 29.7, 26.9, 24.4, 22.4, 17.6 and 15.3 to the protonated carbons HCO, C-2′,
C-6′, C-5′, C-4′, Me-9′, Me-8′, C-1′, Me-(C-3′), Me-(C-6), respectively, while those observed in
the HMBC spectrum allowed to assign the quaternary carbons. Thus, the signals at 162.2,
157.4, 139.7, 136.8, 115.7, 115.4, 114.6 and 75.2 were assigned to C-2, C-4, C-6, C-3′, C-1, C-3,
C-5 and C-7′, respectively. Furthermore, its ESI MS spectrum showed the sodium clusters at
m/z 381 and 379 and the protonated forms at m/z 359 and 357 consistent with the presence
of a chlorine atom.
Compound 1 was isolated for the first time in 1973 from culture filtrate of C. nicotianae,
a pathogenic fungus that induce tobacco anthracnose (Kosuge et al. 1973) and recently from
the culture filtrate of C. higginsianum (Masi et al. 2017). Its total synthesis was realised on
Figure 1. structures of colletochlorin a (1), its 6′-O-S- and 6′-O-R-MtPa esters (5 and 6), orcinol (2), tyrosol
(3) and 1,3-O,O′-diacetylorcinol (4).

4
M. MASI ET AL.
Table 1. 1h and 13c NMr data and hMBc of colletochlorin a (1) and 1h NMr data of Its Mosher’s deriva-
tives (5 and 6).a,b
1
5
6
c
δH (J in Hz)d
HMBC
δH (J in Hz)
δH (J in Hz)
Position
δC
1
2
3
4
5
6
115.7 s
162.2 s
115.4 s
157.4 s
114.6 s
139.7 s
15.3 q
Me-6
h2-1′, hco
h2-1′
h2-1′
Me-6
Me-6
Me-(c-6)
2.60 (3h) s
2.660 (3h) s
3.543 (2h) d (7.5)
5.346 (1h) t (7.5)
2.656 (3h) s
3.541 (2h) d (7.5)
5.342 (1h) t (7.5)
1′
2′
3′
22.4 t
121.6 d
136.8 s
3.40 (2h) d (7.5)
5.27 (1h) t (7.5)
h-2′
h2-1′, Me-(c-3′)
h2-4′, h2-5′, h2-1′,
Me-(c-3′), h-2′
h2-5′
4′
5′
29.7 t
35.9 t
1.32–1.73 (2h) m
2.01–2.43 (2h) m
2.011 (2h) m
2.347 (2h) m
2.008 (2h) m
2.343 (2h) m
h2-4′, h-6′, h-2′,
Me-(c-3’)
6′
79.2 d
3.32 (1h) dd (11.1,
h2-4′, h -5′, Me-8′,
3.724 (1h) dd (11.1,
3.724 (1h) dd (11.1,
3.1)
Me-9′2
3.1)
3.1)
7′
75.2 s
17.6 q
h-6′, Me-8′, Me-9′
h2-4′
Me-9′
Me-8′
Me-(c-3’)
Me-8′
Me-9′
hco
1.80 (3h) s
1.16 (3h) se
1.13 (3h) se
10.13 (1h) s
12.68 (1h) s
2.171 (3h) s
1.138 (3h) s
1.098 (3h) s
10.317 (1h) s
12.528 (1h) s
3.645 (3h) s
2.170 (3h) s
1.194 (3h) s
1.135 (3h) s
10.315 (1h) s
12.523 (1h) s
3.644 (3h) s
24.4 qe
26.9 qe
194.5 d
oh-2
oMe
Ph
7.726–7.368 (5h)
7.713–7.368 (5h)
athe chemical shifts are in δ values (ppm) from tMs.
b2d 1h, 1h (cosY) and 2d 13c, 1h (hsQc) NMr experiments delineated the correlations of all the protons and the correspond-
ing carbons.
cMultiplicities were assigned by dePt spectrum.
dthese data are in according to those previously reported (Kosuge et al. 1974; Mori and sato 1982).
ethe attribution of these signals could be inverted.
1982, but only in racemic mixture (Mori and Sato 1982). Thus, the absolute configuration
(AC) of 1 still remained to be assigned. To reach this goal the AC at C-6’ was determined by
applying the modified Mosher’s method (Ohtani et al. 1991; Cimmino et al. 2017b).
Colletochlorin A was treated with R-(-)-α-methoxy-α-trifluoromethylphenylacetyl (MTPA)
and S-(+)-MTPA chlorides, to convert 1 into the corresponding diastereomeric esters at C-6′
(5 and 6). The spectroscopic data for the S-MPTA and R-MTPA esters (5 and 6, respectively)
were consistent with the structure assigned to 1. The ∆δ (5–6) values for all the protons were
determined as reported in Figure 2, by subtracting the chemical shift of the protons (Table
1) of the 6′-O-S-MTPA (5) from that of 6′-O-R-MTPA (6) esters. The positive ∆δ values were
located on the right-hand side and those with negative values on the left-hand side of model
A as previously reported (Ohtani et al. 1991; Cimmino et al. 2017b). This model allowed to
assign the R configuration at C-6′. The assignment of AC to colletochlorin A represents a key
part in understanding the mode of action of many secondary bioactive metabolites isolated
from microorganisms, plants and marine organisms (Evidente et al. 2011, 2013). In fact the
total syntheses of colletochlorin A, its enantiomer, its bromine and fluorine analogues and
the corresponding colletorin A and its enantiomer are in progress with the aim to evaluate
the structure-activity relationships.

NATURAL PRODUCT RESEARCH
5
Figure 2. structures of 6′-O-S- and 6′-O-R-MtPa of colletochlorin a (5 and 6, respectively), reporting the
Δδ values obtained by comparison (5–6) of each proton system.
Colletochlorin A belong to the class of naturally occurring 2-prenyl orsellinaldehydes
bearing a chlorine attached to the aromatic ring (Kosuge et al. 1973; García-Paión and Collado
2003).
The 1H NMR spectrum of 2 was very similar to that reported in literature for orcinol (Monde
et al. 1998). Its ESI MS spectrum showed the pseudomolecular [M + H]+ ion at m/z 125. To
confirm this structure 2 was also converted into the corresponding 1,3-O,O′-diacetil deriv-
ative (4, Figure 1), previously reported as a synthetic compound (Tangestaninejad et al.
2002). Its 1H NMR spectrum differed from that of 2 mainly for the presence of the singlets of
the two acetyl groups which appeared overlapped at δ 2.30 Its ESI MS spectrum showed the
potassium [M + K]+ and sodium [M + Na]+ clusters at m/z 247 and 231, respectively.
The 1H NMR spectrum of 3 was very similar to that reported in literature for tyrosol (Kimura
and Tamura 1973; Capasso et al. 1992; Cimmino et al. 2017a). Its ESI MS spectrum showed
the dimeric sodiated [2 M + Na]+ form and the sodium cluster [M + Na]+ at m/z 295 and 159,
respectively.
Orcinol (Turner and Aldridge 1983; Monde et al. 1998) and tyrosol (Kimura and Tamura
1973; Capasso et al. 1992) are well known as bioactive fungal and plant metabolites. Tyrosol
was also produced as phytotoxin from fungi pathogenic for some important agrarian crops
(Evidente et al. 2010; Andolfi et al. 2012; Cimmino et al. 2017a) and legumes (Andolfi et al.
2013).
Assayed on punctured leaves, colletochlorin A caused the fast appearance of large necro-
sis on A. artemisiifolia leaves (Figure 3(A)). Tyrosol was weakly active, causing modest necrosis
at the assayed concentration. Orcinol was completely inactive.
Assayed by uptake on ambrosia plantlets, colletochlorin A caused the wilting of the plants
already 24 h after the transfer to water, with the appearance of large necrosis on the leaves
margins, meaning an accumulation of the toxin due to the transpiration (Figure 3(B)).
Conversely, the stem appeared only slightly damaged. In the same assay, orcinol was weakly
active, causing only a slight withering, whereas tyrosol was inactive.
In the assay on Lemna minor, colletochlorin A caused a clear fronds browning, with a total
reduction in chlorophyll content of around 70% (data not shown), whereas the other two
compounds were completely inactive at the concentration tested.

6
M. MASI ET AL.
Figure 3. a: necrosis caused by colletochlorin a (1) on A. artemisiifolia leaves by leaf-puncture assay; B:
wilting of ambrosia plantlet after treatment with colletochlorin a (1) (right) compared with the control
(left).
In the antibiosis assay, colletochlorin A was active only against the gram + bacterium,
Bacillus subtilis, inhibiting its growth as clearly shown by the inhibition halo around the
diskette (data not shown), whereas was inactive against Escherichia coli. The other two com-
pounds were inactive against both bacteria. No toxic effects were visible in the Artemia salina
assay.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured in a MeOH solution on a Jasco P-1010 digital polarimeter
(Tokyo, Japan); 1H and 13C NMR spectra were recorded at 400/100 or 500/125 MHz in CDCl3
on Bruker (Karlsruhe, Germany) and Varian (Palo Alto, CA, USA), spectrometers, respectively,
using the same solvent as internal standard. ESI MS was performed using the LC/MS TOF
system (AGILENT 6230B, HPLC 1260 Infinity, Milan, Italy). Analytical and preparative TLC was
performed on silica gel (Kieselgel 60, F254, 0.25 and 0.5 mm, respectively) and on reversed
phase (Merck Kieselgel 60 RP-18, F254, 0.20 mm) plates (Merck, Darmstadt, Germany). The
spots were visualised by exposure to UV radiation (253), or by spraying first with 10% H2SO4
in MeOH and then with 5% phosphomolybdic acid in EtOH, followed by heating at 110 °C
for 10 min. Column chromatography was performed using silica gel (Merck Kieselgel 60,
0.063–0.200 mm) (Merck).
3.2. Fungal strain
The fungus was isolated from diseased leaves of A. artemisiifolia and identified as C. gloe-
osporioides (Berestetskiy 2016). A monoconidial isolate was deposited in the culture

NATURAL PRODUCT RESEARCH
7
collection of both the All-Russian Research Institute of Plant Protection, Pushkin, Saint-
Petersburg, Russia (MIF 13.14) and the Institute of Sciences of Food Production, Bari, Italy
(ITEM 17400). The isolate was routinely grown and maintained in plates and slants containing
potato-dextrose agar (PDA, Sigma-Aldrich, Chemie Gmbh, Buchs, Switzerland).
3.3. Production, extraction and purification of phytotoxic metabolites
The strain of C. gloeosporioides used in this study was grown on a minimal-defined liquid
medium named M1-D (Pinkerton and Strobel 1976). Small plugs of mycelium obtained by
colonies actively growing on PDA were used to seed 1 L Roux bottles containing 200 mL of
the sterile medium above mentioned. Bottles were kept in still condition at 25 °C in the dark
in an incubator for 4 weeks, then filtered to remove the mycelium and lyophilised. The lyoph-
ilised material, obtained by 11 L of culture filtrates was dissolved under stirring in 600 mL
ultrapure water and extracted with EtOAc (3 × 600 mL). The organic extracts were then com-
bined, dehydrated (Na2SO4), filtered and evaporated under reduced pressure, yielding a dark
oily residue (285 mg). The organic residue was purified by silica gel column chromatography
eluted with CHCl3-i-ProOH (95:5) obtaining 10 groups of homogeneous fractions. The residue
of the fifth fraction (14.3 mg) was further purified by preparative TLC on silica gel eluted with
n-hexane-Me2CO (6:4) yielding colletochlorin A (1, 10.4 mg, 0.94 mg/L) as a homogenous
compound. The residue of the sixth fraction (16 mg) was further purified by preparative TLC
on silica gel eluted with n-hexane-Me2CO (6:4) yielding orcinol (2, 9.4 mg, 0.85 mg/L) as a
homogenous compound. The residue of the eighth fraction (25.7 mg) was further purified
by preparative TLC on silica gel, eluted with CHCl3-i-ProOH (95:5) and by reverse phase TLC,
eluted with Me2CO-H2O (8:2) yielding tyrosol (3, 12.3 mg, 1.11 mg/L) as white solid.
Colletochlorin A (1): amorphous solid; [ꢀ]2D5 + 6.24 (c 0.20) [In Ref. Kosuge et al. 1973:
[ꢀ]2D5 + 11.6 (c 10.0)]; 1H and 13C NMR (400/100 MHz, in CDCl3), are reported in Table 1; ESIMS
(+) m/z: 381 [M + 2 + Na]+, 379 [M + Na]+, 359 [M + 2 + H]+, 357 [M + H]+. These data are in
agreement with those previously reported (Kosuge et al. 1973, 1974; Mori and Sato 1982;
Masi et al. 2017).
Orcinol (2): amorphous solid; 1H NMR (500 MHz, in CDCl3), δ: 6.77 (br s, H-4 and H-6), 6.36
(br s, H-2), 2.33 (s, Me-5). ESIMS (+) m/z: 125 [M + H]+. These data are in agreement with those
previously reported (Monde et al. 1998).
Tyrosol (3): white solid; 1H NMR (500 MHz, in CDCl3), δ: 7.20 (d, J = 8.0 Hz, H-3 and H-5),
6.80 (d, J = 8.0 Hz, H-2 and H-6), 4.90 (s, OH), 3.80 (t, J = 6.4 Hz, H2-2′), 2.80 (t, J = 6.4 Hz, H2-1′).
ESI/MS (+), m/z 295 [2 M + Na]+, 159 [M + Na]+. These data are in agreement with those
previously reported (Kimura and Tamura 1973; Capasso et al. 1992; Cimmino et al. 2017a).
1,3-O,O′-Diacetylorcinol (4). Orcinol (2, 1.4 mg), dissolved in pyridine (20 µL) was acetylated
with acetic anhydride (20 µL) at room temperature. After 24 h the reaction was stopped by
addition of MeOH and the azeotrope formed by addition of C6H6 was dried under reduced
pressure. The residue (1.2 mg) was purified by preparative TLC using CHCl3 as eluent to yield
1,3-O,O′-diacetylorcinol (4, 1.2 mg). Compound 4 had: 1H NMR (500 MHz, in CDCl3), δ: 6.82
(br s, H-4 and H-6), 6.73 (br s, H-2), 2.30 (s, Me-5), 2.19 (s, OAc-1 and OAc-2). ESIMS (+), m/z:
247 [M + K]+ and 231 [M + Na]+.
6′-O-(S)-α-Methoxy-α-trifluoromethyl-α-phenylacetate (MTPA) ester of colletochlorin A
(5). (R)-(-)-MPTA-Cl (20 μL) was added to 1 (1.9 mg) dissolved in dry pyridine (40 μL). The
mixture was kept at room temperature for 1 h and then the reaction was stopped adding

8
M. MASI ET AL.
MeOH and the azeotrope formed by addition of C6H6 was dried under reduced pressure. The
residue (2.1 mg) was purified by preparative TLC, eluted with CHCl3-i-PrOH (98:2), yielding
5 (1.0 mg) as a homogeneous oil. It had: 1H NMR spectrum see Table 1; ESIMS (+) m/z 471
[M + 2 + H]+ and 469 [M + H]+.
6′-O-(R)-α-Methoxy-α-trifluoromethyl-α-phenylacetate (MTPA) ester of colletochlorin A
(6). (S)-(+)-MPTA-Cl (20 μL) was added to 1 (1.9 mg) dissolved in dry pyridine (40 μL). The
reaction was carried out under the same conditions used for preparing 5 from 1. The puri-
fication of the crude residue (2.0 mg) by preparative TLC eluted with CHCl3-i-PrOH (98:2), to
give 6 (0.9 mg) as homogeneous oil. It had: 1H NMR spectrum see Table 1; ESIMS (+) m/z 471
[M + 2 + H]+ and 469 [M + H]+.
3.4. Biological assays
The three metabolites were first dissolved in MeOH and then diluted with distilled water to
the desired concentration.
3.4.1. Leaf-puncture assay
The toxins were tested at 2 μg/μL on leaves of A. artemisiifolia. Droplets (20 μL) of the solution
containing the compounds were applied to young detached leaves previously punctured
with a needle. Five replications were used for each compound. Leaves were kept in a mois-
tened chamber under continuous fluorescent lights at 25 °C. The eventual appearance of
symptoms was observed three days after droplet application. Control treatments were car-
ried out by applying droplets containing only water with the MeOH.
3.4.2. Cut plant assay
Young ambrosia plants (around 1 month old) grown in growth chamber were cut and the
stem (having 5–6 leaves) immersed in the solutions (10−3 M) for 24 h. Plants were then
immersed in water for 48 h before observing symptom appearance.
3.4.3. Lemna minor assay
The compounds were tested on Lemna minor using a protocol previously described (Vesonder
et al. 1992) at a concentration of 2 μg/μL using 100 μL per each pot. Chlorophyll content
was determined as reported and expressed as percentage reduction in comparison to the
untreated fronds (Vurro et al. 2006).
3.4.4. Antibiosis assay
A disc assay on both one Gram+(Bacillus subtilis) and one Gram- (Escherichia coli) bacterium
was carried out by following the procedure already described (Bottalico et al. 1990).
Metabolites were assayed up to 150 μg per diskette.
3.4.5. Zootoxic assay
This assay was performed on Artemia salina (brine shrimps) by following the procedure
already described by Bottalico et al. (1990). A concentration of 70 μg/well was used for each
compound. A negative control was also assayed (solvent in water).

NATURAL PRODUCT RESEARCH
9
4. Conclusions
Three metabolites were isolated for the first time from the culture filtrates of C. gloeosporiodes
and identified as the already known colletochlorin A, orcinol and tyrosol. Among them colle-
tochlorin A was significantly phytotoxic against the host plant A. artmisiifolia showing also
activity against L. minor associated to only inhibitory activity against gram + bacteria.
Colletochlorin A was only recently reported, from some of the authors, for its modest phy-
totoxic activity against Sonchus arvevsis (Masi et al. 2017). Considering the lack of zootoxicity
and the activity against gram- bacteria, colletochlorin A appears to be a potential bioherbi-
cide against A. artemisiifolia. Thus, the development of its total enatioselective synthesis,
now in progress, represents an alternative for its large-scale preparation in respect to the
fermentative production.
Acknowledgements
The work was carried out in the frame of the COST Action FA1203 “Sustainable management of
Ambrosia artemisiifolia in Europe”(SMARTER). Prof. Antonio Evidente is associated with the Istituto di
Chimica Biomolecolare del CNR, Pozzuoli, Italy.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by grants of Department of Chemical Science, University of Naples Federico
II, Napoli, Italy.
ORCID
Marco Masi
http://orcid.org/0000-0003-0609-8902
Alessio Cimmino
Maurizio Vurro
Antonio Evidente
http://orcid.org/0000-0002-1551-4237
http://orcid.org/0000-0001-6875-4093
http://orcid.org/0000-0001-9110-1656
References
Andolfi A, Maddau L, Cimmino A, Linaldeddu BT, Franceschini A, Serra S, Basso S, Melck D, Evidente
A. 2012. Cyclobotryoxide, a phytotoxic metabolite produced by the plurivorous pathogen
Neofusicoccum australe. Journal of Natural Products. 75:1785–1791.
Andolfi A, Cimmino A, Villegas-Fernández AM, Tuzi A, Santini A, Melck D, Rubiales D, Evidente A. 2013.
Lentisone, a new phytotoxic anthraquinone produced by Ascochyta lentis, the causal agent of
Ascochyta blight in Lens culinaris. J Agric Food Chem. 61:7301–7308.
Berestetskiy AO. 2016. Biological activity of extracts from different pathogens of common ragweed.
Proceedings of 7th International Weed Science Congress“Weed Science and Management to Feed
the Planet”; Jun 19–25, Prague, p. 139.
Berger S, Braun S. 2004. 200 and more basic NMR experiments: A practical course. 1st ed. Weinheim:
Wiley-VCH.
Bottalico A, Capasso R, Evidente A, Randazzo G, Vurro M. 1990. Cytochalasins: Structure-activity
relationships. Phytochemistry. 29:93–96.

10
M. MASI ET AL.
Boyette CD, JrPC Quimby, Bryson CT, Egley GH, Fulgham FE. 1993. Biological control of hemp sesbania
(Sesbania exaltata) under field conditions with Colletotrichum truncatum formulated in an emulsifiable
invert. Weed Sci. 41:496–499.
Capasso R, Cristinzio G, Evidente A, Scognamiglio F. 1992. Isolation, spectroscopy and selective phytoxic
effects of polyphenols from vegetable waste waters. Phytochemistry. 31:4125–4128.
Cimmino A, Masi M, Evidente M, Superchi S, Evidente A. 2015a. Fungal phytotoxins with potential
herbicidal activity: chemical and biological characterization. Nat Prod Rep. 32:1629–1653.
Cimmino A, Mathieu V, Masi M, Baroncelli R, Boari A, Pescitelli G, Federin M, Lisy R, Evidente M, Zonno
MC, et al. 2015b. Higginsianins A and B, two diterpenoid α-pyrones produced by Colletotrichum
higginsianum, with in vitro cytostatic activity. J Nat Prod. 79:116–125.
Cimmino A, Cinelli T, Masi M, Reveglia P, da Silva MA, Mugnai L, Michereff SJ, Surico G, Evidente A.
2017a. Phytotoxic lipophylic metabolites produced by grapevine strains of Lasiodiplodia species in
Brazil. J Agric Food Chem. 65:1102–1107.
Cimmino A, Masi M, Evidente M, Superchi S, Evidente A. 2017. Application of Mosher’s method for
absolute configuration assignment to bioactive plants and fungi metabolites. J Pharmaceut Biomed.
144:59–89. doi:10.1016/j.jpba.2017.02.037.
Committee on Mycoherbicides for Eradicating Illicit Drug Crops. 2011. Biological control of undesirable
plants. In National Research Council (USA) editor. Feasibility of using mycoherbicides for controlling
illicit drug crops. Washington, DC: Board on Agriculture and Natural Resources, National Research
Council (USA), The National Academies Press; p. 27–46.
Essl F, Biró K, Brandes D, Broennimann O, Bullock J, Chapman D, Chauvel B, Dullinger S, Fumanal B,
Guisan A, et al. 2015. Biological flora of the british isles: Ambrosia artemisiifolia. Journal of Ecology.
103:1069–1098.
Evidente A, Punzo B, Andolfi A, Cimmino A, Melck D, Luque J. 2010. Lipophilic phytotoxins produced
by Neofusicoccum parvum, a grapevine canker agent. Phytopathol Mediterr. 49:74–79.
Evidente A, Andolfi A, Cimmino A. 2011. Relationships between the stereochemistry and biological
activity of fungal phytotoxins. Chirality. 23:674–693.
Evidente A, Cimmino A, Andolfi A. 2013. The effect of stereochemistry on the biological activity of
natural phytotoxins, fungicides, insecticides and herbicides. Chirality. 25:59–78.
García-Paión CM, Collado IG. 2003. Secondary metabolites isolated from Colletotrichum species. Nat
Prod Rep. 20:426–431.
Gerber E, Schaffner U, Gassmann A, Hinz HL, SEIER M, Müller-Schärer H. 2011. Prospects for biological
control of Ambrosia artemisiifolia in Europe: learning from the past. Weed Res. 51:559–573.
KimuraY, Tamura S. 1973. Isolation of l -β-phenyllactic acid and tyrosol as plant growth regulators from
Gloeosporium laeticolor. Agric Biol Chem. 37:2925–2925.
KosugeY, Suzuki A, Hirata S, Tamura S. 1973. Structure of colletochlorin from Colletotrichum nicotianae.
Agr Biol Chem. 37:455–456.
Kosuge Y, Suzuki A, Tamura S. 1974. Structures of colletochlorin C, colletorin A and colletorin C from
Colletotrichum nicotianae. Agr Biol Chem. 38:1265–1267.
Masi M, Cimmino A, Boari A, Tuzi A, Zonno MC, Baroncelli R, Vurro M, Evidente A. 2017. Colletochlorins
E and F, new phytotoxic tetrasubstituted pyran-2-one and Dihydrobenzofuran, isolated from
Colletotrichum higginsianum with potential herbicidal activity. J Agric Food Chem. 65:1124–1130.
Monde K, Satoh H, Nakamura M, Tamura M, Takasugi M. 1998. Organochlorine compounds from a
terrestrial higher plant: structures and origin of chlorinated orcinol derivatives from diseased bulbs
of Lilium maximowiczii. J Nat Prod. 61:913–921.
Mori K, Sato K. 1982. A general synthetic method for prenylated phenols of microbial origin: Synthesis
of colletochlorins A and B. Tetrahedron. 38:1221–1225.
Ohtani I, Kusumi T, Kashman Y, Kakisawa H. 1991. High-field FT NMR application of Mosher’s method.
The absolute configurations of marine terpenoids. J Am Chem Soc. 113:4092–4096.
Phoulivong S, Cai L, Chen H, McKenzie EHC, Abdelsalam K, Chukeatirote E, Hyde KD. 2010. Colletotrichum
gloeosporioides is not a common pathogen on tropical fruits. Fungal Divers. 44:33–43.
Pinkerton F, Strobel GA. 1976. Serinol as an activator of toxin production in attenuated cultures of
Helminthosporium sacchari. Proc Natl Acad Sci USA. 73:4007–4011.

NATURAL PRODUCT RESEARCH
11
Sutton BC. 1992. The genus Glomerella and its anamorph Colletotrichum. In Bailey JA, Jeger MJ, editors.
Colletotrichum, biology, pathlogy and control. Wallingford: CAB International; p. 1–26.
Tangestaninejad S, Habibi MH, Mirkhani V, Moghadam M. 2002. Rapid and efficient acetylation of
alcohols and phenols with acetic anhydride using tin (IV) porphyrin as catalyst. Synth Commun.
32:1337–1343.
Turner WB, Aldridge DC. 1983. Fungal Metabolites II. London: Academic Press.
Vesonder RF, Labeda DP, Peterson RE. 1992. Phytotoxic activity of selected water-soluble metabolites
of Fusarium against Lemna minor L. (Duckweed). Mycopathologia. 118:185–189.
Vurro M, Boari A, Pilgeram AL, Sands DC. 2006. Exogenous amino acids inhibit seed germination and
tubercle formation by Orobanche ramosa (Broomrape): Potential application for management of
parasitic weeds. Biol Control. 36:258.
Wymore LA, Poirier C, Watson AK, Gotlieb AR. 1988. Colletotrichum coccodes, a potential bioherbicide
for control of velvet leaf (Abutilon theophrasti). Plant Dis. 72:534–538.
Zonno MC, Cimmino A, Vurro M, Evidente A. 2015. Effects of fungal toxins on Ambrosia artemisiifolia.
Proceedings of 17th European Weed Research Society Symposium‘Weed management in changing
environments’; Jun 23–26, Montpellier, France, p. 34.
Zonno MC, Vurro M, Boari A, Cimmino A, Masi M, Reveglia P, Berestetskiy A, Evidente A. 2016. Phytotoxic
metabolites produced by Colletotrichum gloeosporioides as potential natural herbicides for the
control of Ambrosia artemisiifolia. Proceedings of 7th International Weed Science Congress; Jun
19–25, Prague, p. 464.