Insights on the susceptibility of plant pathogenic fungi to phenazine-1-carboxylic acid and its chemical derivatives

Article abstract of DOI:10.1080/14786419.2012.696257

Pseudomonas chlororaphis subsp. aureofaciens strain M71 produced two phenazine compounds as main secondary metabolites. These metabolites were identified as phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH P). In this study, the spectrum of the activity of PCA and 2-OH P was evaluated against a group of crop and forestal plant pathogenic fungi by an agar plate bioassay. PCA was active against most of the tested plant pathogens, while 2-OH P slightly inhibited a few fungal species. Furthermore, four semisynthesised derivatives of PCA (phenazine-1-carboxymethyl, phenazine-1-carboxamide, phenazine-1-hydroxymethyl and phenazine-1-acetoxymethyl) were assayed for their antifungal activity against 11 phytopathogenic species. Results showed that the carboxyl group is a structural feature important for the antifungal activity of PCA. Since the activity of phenazine-1-carboxymethyl and phenazine-1- carboxamide, the two more lipophilic and reversible PCA derivatives remained substantially unaltered compared with PCA.

Full text of DOI:10.1080/14786419.2012.696257

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Insights on the susceptibility of plant
pathogenic fungi to phenazine-1-
carboxylic acid and its chemical
derivatives
Gerardo Puopolo a , Marco Masi b , Aida Raio c , Anna Andolfi b
Astolfo Zoina a , Alessio Cimmino b & Antonio Evidente b
,
a Dipartimento di Arboricoltura, Botanica e Patologia Vegetale ,
Universitá di Napoli Federico II , Via Università 100, 80055
Portici , Italy
b Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente
e delle Produzioni Animali , Università di Napoli Federico II , Via
Università 100, 80055 Portici , Italy
c Istituto per la Protezione delle Piante, CNR-Area di Ricerca di
Firenze , 50019 Sesto Fiorentino , Italy
Published online: 25 Jun 2012.
To cite this article: Gerardo Puopolo , Marco Masi , Aida Raio , Anna Andolfi , Astolfo Zoina ,
Alessio Cimmino & Antonio Evidente (2013): Insights on the susceptibility of plant pathogenic fungi
to phenazine-1-carboxylic acid and its chemical derivatives, Natural Product Research: Formerly
Natural Product Letters, 27:11, 956-966
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Natural Product Research, 2013
Vol. 27, No. 11, 956–966, http://dx.doi.org/10.1080/14786419.2012.696257
Insights on the susceptibility of plant pathogenic fungi to phenazine-
1-carboxylic acid and its chemical derivatives
Gerardo Puopoloa, Marco Masib, Aida Raioc, Anna Andolfib, Astolfo Zoinaa,
Alessio Cimminob and Antonio Evidenteb
*
a
´
Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Universita di Napoli Federico II,
Via Universita 100, 80055 Portici, Italy; Dipartimento di Scienze del Suolo, della Pianta,
b
`
`
`
dell’Ambiente e delle Produzioni Animali, Universita di Napoli Federico II, Via Universita 100,
80055 Portici, Italy; Istituto per la Protezione delle Piante, CNR-Area di Ricerca di Firenze,
c
50019 Sesto Fiorentino, Italy
(Received 28 November 2011; final version received 6 April 2012)
Pseudomonas chlororaphis subsp. aureofaciens strain M71 produced two phena-
zine compounds as main secondary metabolites. These metabolites were identified
as phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH P). In this
study, the spectrum of the activity of PCA and 2-OH P was evaluated against a
group of crop and forestal plant pathogenic fungi by an agar plate bioassay. PCA
was active against most of the tested plant pathogens, while 2-OH P slightly
inhibited a few fungal species. Furthermore, four semisynthesised derivatives of
PCA (phenazine-1-carboxymethyl, phenazine-1-carboxamide, phenazine-
1-hydroxymethyl and phenazine-1-acetoxymethyl) were assayed for their anti-
fungal activity against 11 phytopathogenic species. Results showed that the
carboxyl group is a structural feature important for the antifungal activity of
PCA. Since the activity of phenazine-1-carboxymethyl and phenazine-1-carbox-
amide, the two more lipophilic and reversible PCA derivatives remained
substantially unaltered compared with PCA.
Keywords: Pseudomonas chlororaphis subsp. aureofaciens; phenazine derivatives;
antifungal activity
1. Introduction
Phenazines are a large group of nitrogen-containing heterocyclic compounds produced by
members of Pseudomonas, Streptomyces and a few other bacterial genera from soil or
marine habitats (Abken et al., 1998; Laursen & Nielsen, 2004). These compounds showed
an interesting biological activity that prompted the development of synthetic analogues
with antimicrobial, antitumor, antimalaria and antiparasitic activities. More than 100
phenazines of natural origin and 6000 synthetic phenazines have been reported during the
last century (Laursen & Nielsen, 2004; L.S. Pierson & E.A. Pierson, 2010). Natural
phenazines are generally simple functionalised phenazine derivatives. They include
terpenoid phenazines, phenazines derived from both saphenic and griseoluteic acids and
others. Synthetic phenazines include phenazine-1-carboxamide, dimeric phenazines,
*Corresponding author. Email: evidente@unina.it
© 2013 Taylor & Francis

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phenazine-6,11-dione, phenazine-5,10-dioxide, benzophenazine-5-sulfonic acid and ana-
logues (Cimmino et al., 2012)
The genus Pseudomonas contains a number of species that share traits that can be
exploited for plant protection. They produce different kinds of antimicrobial compounds
with a wide spectrum of activity, such as the phenazines (Weller, 2007). In particular,
members of the species P. aeruginosa, P. fluorescens and P. chlororaphis are able to
produce these phenazine antibiotic compounds that subsequently represent the main
secondary metabolites responsible for the biological control of important plant pathogens,
namely Gaeumannomyces graminis var. tritici, Fusarium oxysporum f.sp. radicis-lycopersici
and Rhizoctonia solani (Chin-A-Woeng et al., 1998; Liu et al., 2007; Shanmugaiah,
Mathivanan, & Varghese, 2010; Thomashow, Weller, Bonsall, & Pierson, 1990).
Recently, Raio et al. (2011) reported that P. chlororaphis subsp. aureofaciens strain
M71 showed to be a potential agent for the control of Seiridium cardinale, the causative
agent of cypress (Cupressus sempervirens L.) bark canker disease. Production of phenazine-
1-carboxylic acid (PCA, 1, Figure 1) by strain M71 involved the control of this plant
pathogen. This compound was able to inhibit the in vitro growth of S. cardinale and other
cypress pathogenic fungi, such as Diplodia cupressi, Seiridium cupressi and Seiridium
unicorne, while the 2-hydroxyphenazine (2-OH P, 2, Figure 1) the second phenazine
isolated from the culture filtrates of strain M71 lacked this activity (Raio et al., 2011).
Although several works have been documented so far on the importance of the
phenazines in the biological control of phytopathogenic fungi, little is known on how their
activity can vary against different fungal species. PCA extracted from P. fluorescens strain
2-79 was screened against several fungi and bacteria and revealed the greatest inhibitory
activity against G. graminis var. tritici and Pythium spp. (Gurusiddaiah, Weller, Sarkar, &
Cook, 1986). Subsequently, Mazzola, Fujimoto, Thomashow, and Cook (1995) evidenced
that some G. graminis var. tritici isolates were able to tolerate PCA at a concentration of
1 mg mLꢀ1, but so far nothing is known on the effect of PCA on other plant pathogenic
fungi, especially on fungal species that attack forestry plants.
Therefore, the first aim of this work was to assess the susceptibility of a group of fungi
pathogenic to crop and forestal plants towards PCA and 2-OH P produced by
P. chlororaphis subsp. aureofaciens strain M71. A further aim was to define the structural
features involved in the antifungal activity of PCA, assayed in comparison with four
semisynthetic derivatives.
COR
N
OH
10a
4a
9a
5a
N
9
1
4
2
10
8
7
2
3
5
N
6
N
2
1 R=OH
3 R=OMe
4 R=NH2
CH2OR
N
1
N
5 R=H
6 R=Ac
Figure 1. Structures of PCA (1) and its derivatives (3–6) and that of 2-OH P (2).

958
G. Puopolo et al.
2. Results and discussion
PCA and 2-OH P (respectively, 1 and 2 in Figure 1) were evaluated for their antifungal
activity against several plant pathogenic fungi at concentrations of 10, 25 and 50 mg mLꢀ1
(Table 1). PCA was the most effective in the inhibition of the major part of the plant
pathogenic fungi evaluated in this work (Table 1). Moreover, these results are in total
agreement with previously published data that reported phenazine-producing
Pseudomonas species as biocontrol agents of plant pathogenic fungi (Pierson &
Thomashow, 1992; Raio et al., 2011; Vincent et al., 1991). On the contrary, 2-OH P did
not affect the growth of 15 out of the 25 tested fungal species. Four fungi were slightly
inhibited (less than 10% reduction in colony radial growth), while the colony development
of four Fusarium species and of G. graminis var. tritici was reduced between 18% and 34%
at a concentration of 50 mg mLꢀ1 (Table 1).
These data lead us to hypothesise that the production of 2-OH P in strain M71 could
be related to some other biological mechanisms. Recently, Maddula, E.A. Pierson, and
L.S. Pierson (2008) showed that some mutant derivatives of P. chlororaphis strain 30-84,
that produced only PCA or overproduced 2-hydroxy-phenazine-1-carboxylic acid (2-OH
PCA), were altered in their ability to form biofilms and inhibit G. graminis var. tritici.
Thus, these evidences encourage further investigations on the role played by 2-OH P in the
ecology of strain M71.
Each fungal species showed a different sensitivity to PCA, even though increasing
concentration of the compound in the medium was always associated with a strong
reduction of colony growth, with the only exception of R. solani. PCA was able to totally
inhibit the growth of Phytophthora nicotianae and G. graminis var. tritici at concentrations
of 25 and 50 mg mLꢀ1. Moreover, P. cinnamomi and Pythium ultimum grew on PDA plates
amended with PCA 25 mg mLꢀ1 while P. nicotianae and P. cambivora were strongly
inhibited at this concentration (Table 1). P. cinnamomi, P. ultimum and R. solani overgrew
on the PDA plates amended with 25 mg mLꢀ1 PCA, but it was observed that their mycelia
were thinner than the group (data not shown). In a follow-up experiment carried out in
liquid media, each of these three fungal species exhibited a slower growth rate than the
group when grown in a tube containing PDB amended with 25 mg mLꢀ1 of PCA (Figure 2).
At the end of this second experiment, organic extracts of the broth media were analysed by
TLC for the presence of PCA degradation products. For the three fungal species
mentioned above, no PCA degradation products were detected. The absence of
degradation products leads us to hypothesise that P. cinnamomi, P. ultimum and R.
solani share some biological mechanism that allows them to tolerate PCA. One of these
mechanisms might be represented by ATP-binding cassette (ABC) transporters. The
activity of BcatrB (Stefanato et al., 2009) in Botrytis cinerea provides protection against
PCA and phenazine-1-carboxyamide when these compounds were present with the fungus
on tomato leaves at the same time (Schoonbeek, Raaijmakers, & De Waard, 2002).
These results and those reported by Raio et al. (2011) strongly suggest that PCA is
involved in the antagonistic activity of strain M71. Since fungi tested in this work showed a
variable sensitivity to this antibiotic compound (Table 1 and Figure 2), a study on the
relationship between the structure and antifungal activity of PCA was carried out to
further investigate the chemical features of PCA associated with its antifungal activity.
This study was carried out through the preparation of semisynthetic derivatives
from PCA.
PCA (1) was converted into four derivatives by chemical modification of the carboxyl
group. In fact, by the reaction with diazomethane, 1 was quantitatively converted into the
corresponding methyl ester (3, Figure 1), a reversible derivative (Axel, 1983), which, in

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959

960
G. Puopolo et al.
PDB
PDB+PCA
100
***
75
50
25
0
***
**
Phytophthora cinnamomi
Pythium ultimum
Rhizoctonia solani
Figure 2. Effects of PCA at 25 mg mLꢀ1 on the mycelium dry weight of three phytopathogenic
microorganisms.
Notes: Differences between means were compared using Student’s t test. *** p 5 0.001 ** p 5 0.1.
turn was converted into the corresponding amide (4, Figure 1), another reversible
derivative, by nucleophilic acyl substitution using ammonia (Chin-A-Woeng et al., 1998).
Furthermore, the LiAlH4 reduction of the methyl ester (3) yielded 1-hydromethylphena-
zine (5, Figure 1) (L. Birkhofer & A. Birkhofer, 1952; Ichiro & Kan, 1964), which showed
a permanent modification of the carboxyl group. This latter derivative was converted into
the corresponding acetyl derivative (6, Figure 1) (Ichiro & Kan, 1964), in which the
primary hydroxy group was reversibly modified. The 3–6 derivative structures confirmed
1
by spectroscopic investigation and some data, essentially H NMR and ESI, are reported
here for the first time.
A group of 11 microorganisms including tolerant isolates (i.e. R. solani), highly
sensitive (i.e. G. graminis var. tritici) and slightly sensitive to PCA (A. alternata) at
25 mg mLꢀ1 were selected among the 25 species considered previously in order to evaluate
the antifungal activity of the derivatives 3–6 compared with the parent PCA (Table 2).
PCA proved to be the most effective compound against B. cinerea, F. oxysporum f.sp.
radicis-lycopersici, G. graminis var. tritici, Ophiostoma novo-ulmi, P. cambivora and S.
cardinale. At the same time, P. ultimum, was not affected by any derivatives while A.
alternata was inhibited by all derivatives at a higher percentage than PCA.
A remarkable reduction or the total loss of antifungal activity was observed for nine
isolates in the case of the hydroxymethyl and its corresponding acetyl derivative (5 and 6).
This effect was probably due to the reduction of the carboxyl group to a primary hydroxy
group (Table 2), outlining that the carboxyl group is associated with the inhibitory activity
of PCA against different fungal species. This is also supported by the comparison of PCA
and 2-OH P activities.
Interestingly, corresponding reversible methyl ester and amide derivatives (3 and 4)
showed an antifungal activity similar to PCA when tested against G. graminis var. tritici.
Probably, derivatives 3 and 4 were enzymatically hydrolysed into PCA by the fungus
according to a well-known mechanism that is frequently called ‘lethal metabolism’
(Kamrin, 1990). In comparison with PCA, a remarkable reduction in the inhibitory

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961
Table 2. Susceptibility of 11 phytopathogenic fungi and oomycetes to PCA (1) and its derivatives
(3–6).
Compounds
Phytopathogenic fungal strains and oomycetes
1a
3
4
5
6
Alternaria alternata
Botrytis cinerea
30db
41a
82a
100a
53b
63ab
5c
0a
0c
31a
29b
53a
18c
37e
98ab
100a
49b
5c
0a
36a
0c
35d
30b
69b
92b
70ab
77a
29a
0a
47b
10d
57c
74c
20c
21c
0c
0a
0c
0c
33b
38c
14cd
43d
37d
21c
14c
0c
0a
26b
0c
Fusarium oxysporum f.sp. radicis-lycopersici
Gaeumannomyces graminis var. tritici
Ophiostoma novo-ulmi
Phytophthora cambivora
Phytophthora cinnamomi
Pythium ultimum
Rhizoctonia solani
Sclerotinia sclerotiorum
Seiridium cardinale
26b
13b
52a
32b
27b
Notes: Treatments with the same letters are not statistically different according to Tukey’s test
(p 5 0.01)
a1: phenazine-1-carboxylic acid; 3: phenazine-1-carboxymethyl; 4: phenazine-1-carboxamide;
5: phenazine-1-hydroxymethyl; 6: phenazine-1-acetoxymethyl.
bThe reduction of radial mycelial growth expressed as a percentage.
activity was recorded for derivatives 3 and 4 against F. oxysporum f.sp. radicis-lycopersici
and B. cinerea (Table 2). Total growth inhibition of O. novo-ulmi was observed in the
presence of compound 3, which also proved to be the most effective derivative against A.
alternata and R. solani. The two Phytophthora species were more sensitive to derivative 4
(Table 2). These results showed that the carboxyl group is a structural feature important
for the antifungal activity of PCA.
It is well known that phytopathogenic fungi have developed several mechanisms that
allow them to counteract the effects of antibiotic compounds produced by biocontrol
agents. These responses include different defensive mechanisms (Duffy, Schouten, &
Raaijmakers 2003). So far, the interaction between plant pathogenic fungi and phenazines
has not been broadly investigated. This study gives the opportunity to formulate some
hypotheses on the response of some pathogenic fungi to PCA. For instance, the data
presented in this work lead us to hypothesise that R. solani has evolved a mechanism more
specific for the detoxification of PCA than for the compounds 3 and 4. Furthermore, the
partial loss of activity against F. oxysporum f.sp. radicis-lycopersici could be associated
with a particular function that PCA plays inside the fungal host cells.
3. Experimental
3.1. General
IR Spectra: Perkin-Elmer (Norwalk, CT) Spectrum One FT-IR spectrometer; neat, ꢀ in
cmꢀ1. UV Spectra: Perkin-Elmer Lambda 25 UV/Visible spectrophotometer in MeOH;
1
ꢁmax (log ") in nm. H NMR Spectra: Bruker (Karlsruhe, Germany) spectrometer at 400
and 600 MHz; ꢂ in ppm relative to each solvent used as internal standard, J in Hz.
ESI-MS: Agilent Quadrupole 6120 LC/MS (Waghaeusel-Wiesental, Germany)
spectrometer; in m/z. Anal. and prep. TLC: silica gel (SiO2; Kieselgel 60, F254, 0.25 and
0.50 mm, respectively, Merck, Darmstadt, Germany) plates; the spots were visualised by
exposure to UV radiation (254 nm) and/or by spraying first with 10% H2SO4 in methanol

962
G. Puopolo et al.
and then with 5% phosphomolybdic acid in ethanol, followed by heating at
110ꢁC for 10 min. Column chromatography (CC): SiO2 column (Merck, Kieselgel 60,
0.063–0.200 mm).
3.2. Bacterial strain and fungal isolates
Pseudomonas chlororaphis subsp. aureofaciens strain M71, a bacterium isolated from
tomato rhizosphere (Puopolo, Raio, Pierson, & Zoina, 2011), was deposited in the
herbarium of the Dipartimento di Arboricoltura, Botanica e Patologia Vegetale Universita
´
di Napoli Federico II, Portici, Italy. The bacterial strain was maintained in 40% glycerol
at ꢀ20ꢁC and was routinely grown on Nutrient Agar plates. Fungal isolates employed in
this work belong to the collection of the Dipartimento di Arboricoltura, Botanica e
Patologia Vegetale Universita di Napoli Federico II, Portici, Italy, and of the Istituto per
´
la Protezione delle Piante, CNR-Area di Ricerca di Firenze. Fungal isolates were stored in
sterile tubes containing potato dextrose agar (PDA; Difco, Detroit, MI) and routinely
grown on PDA plates.
3.3. Production, extraction and purification of phenazines
Strain M71 was grown for 1 week at 27ꢁC in 4 L of PPMD, as previously reported (Wood,
Gong, Daykin, Williams, & Pierson, 1997). The lyophilised material from the culture
filtrates was dissolved in distilled water, acidified to pH 2 with concentrated HCl and
extracted exhaustively with CHCl3 (3 ꢂ 200 mL). The organic extracts were combined,
dried with Na2SO4, filtered and evaporated under reduced pressure. The brown oily
residue (485.7 mg) was fractionated by CC on SiO2 eluted with CHCl3-i-PrOH 9:1 yielding
four groups of homogeneous fractions. The residue of the first fraction yielded the main
compound, identified as PCA (1), a homogeneous compound (Rf ¼ 0.69, 106.5 mg).
The residue of the second fraction (250 mg) was further purified by CC on SiO2 eluted with
CHCl3-i-PrOH 95:5 yielding three groups of homogeneous fractions. The residue of the
first fraction yielded a further amount (18.6 mg) of the main compound (1) as a
homogeneous compound. The residue of the second and third fractions, which
proved to be a mixture of two metabolites, was combined and further purified by prep.
TLC (SiO2; eluent CHCl3-i-PrOH 95:5) to give 16 mg of 1 (total 141.1 mg, 35.2 mg Lꢀ1
)
and another homogeneous compound (64 mg), identified as 2-OH P (2, Rf ¼ 0.35,
16 mg Lꢀ1).
3.3.1. Phenazine-1-carboxylic acid (1)
IR: 2916, 1718, 1622, 1600, 1562, 1522 (Brisbane, Janik, Tate, & Warren, 1987): IR at
0.5% KCl: 3040, 3020, 1740, 1625, 1605, 1565, 1525); UV: 364 (3.81). 1H NMR (recorded
in CDCl3) and ESI-MS were previously reported (Raio et al., 2011).
3.3.2. 2-Hydroxyphenazine (2)
IR: 3328, 1647,1449,1411; UV: 5220 (Pierson & Thomashow, 1992: UV/Visible, 30%
CH3CN: 372, 250). 1H NMR (recorded in CD3OD) and ESI-MS were previously reported
(Raio et al., 2011).
3.3.3. Phenazine-1-carboxymethyl (3)
An ethereal solution of CH2N2 (300 mL) was added to PCA (1, 60 mg) dissolved in MeOH
(2 mL). The reaction was carried out overnight at room temperature (r.t.) in the dark. The
reaction was stopped by evaporation under a N2 stream. The residue (63.2 mg) was

Natural Product Research
963
purified by prep. TLC (SiO2; eluent CHCl3-i-PrOH 95:5) to obtain 1-carboxymethylphe-
1
nazine (3, 50 mg). IR: 1718, 1559, 1521, 1462; UV: 365 (4.12), 248 (4.82); H NMR,
CD3OD, ꢂ: 8.39 (d, J ¼ 8.7, H-4), 8.34 (d, J ¼ 6, H-2), 8.33 (d, J ¼ 8.7, H-6), 8.23 (d,
J ¼ 6.4, H-9), 7.87 (m, H-3, H-7 and H-8), 4.11 (s, OMe); ESI-MS (þ): 261 [M þ Na]þ, 239
[M þ H]þ.
3.3.4. Phenazine-1-carboxamide (4)
To phenazine-1-carboxymethyl (3, 15.2 mg) dissolved in MeOH (2 mL), 30% NH3 (10 mL)
was added. The reaction was carried out at r.t. overnight. After 24 h, the reaction was
complete and the mixture was evaporated under reduced pressure. The residual crude oil
(14.8 mg) was purified by prep. TLC (SiO2, eluent CHCl3-i-PrOH 95:5) to yield derivative
4 (11.4 mg) as a homogeneous compound. IR: 3327, 1694, 1524; UV: 366 (4.51), 248 (4.97);
1H NMR (recorded in CDCl3) is very similar to that previously reported (Chin-A-Woeng
et al., 1998); ESI-MS (þ): 246 [M þ Na]þ, 224 [M þ H]þ.
3.3.5. Phenazine-1-hydroxymethyl (5)
LiAlH4 (9 mg) was added to phenazine-1-carboxymethyl (3, 26 mg) dissolved in anhydrous
ethyl ether (2 mL) by stirring with ice. After 30 min, 2 mL of EtOAc and 8 mL of distilled
H2O were added. The solution was neutralised with 1 M HCl and then extracted with
EtOAc (3 ꢂ 20 mL). The organic extracts were combined, dried (Na2SO4) and evaporated
under reduced pressure. The oily residue (24.2 mg) was purified by prep. TLC (SiO2, eluent
CHCl3-i-PrOH 9:1) to yield derivative 5 (17.2 mg). IR: 3280, 11524, 1425; UV: 362 (4.17),
1
250 (5.05). H NMR, CDCl3, ꢂ: 8.32 (dd, J ¼ 7.4, 2.2, H-4), 8.25 (dd, J ¼ 7.0, 2.2, H-2),
8.15 (d, J ¼ 8.7, H-6), 8.02 (d, J ¼ 6.8, H-9), 7.96 (m, H-3, H-7 and H-8), 4.89 (s, CH2-OH);
ESI-MS (þ): 233 [M þ Na]þ, 211 [M þ H]þ.
3.3.6. Phenazine-1-acetoxymethyl (6)
To phenazine-1-hydroxymethyl (8.2 mg) dissolved in pyridine (240 mL), Ac2O (240 mL) was
added under stirring. The reaction was carried out at 80ꢁC for 30 min. The reaction was
stopped by the addition of MeOH and evaporation of the mixture by a N2 stream. The
residual crude oil (7.2 mg) was purified by prep. TLC (SiO2, eluent CHCl3-i-PrOH 9:1) to
yield derivative 6 (6.9 mg) as a homogeneous compound. IR: 1744, 1526, 1463, 1228
1
(cmꢀ1): 1742, 1242); UV: 363 (4.06), 250 (4.76); H NMR,
nujol
max
((Ichiro & Kan, 1964): ꢀ
CDCl3, ꢂ: 8.50 (br d, J ¼ 7.4, H-4), 8.46 (br d, J ¼ 7, H-6), 8.36 (d, J ¼ 8.4, H-9), 7.95 (m,
H-2, H-3, H-7, H-8), 6.00 (s, CH2O), 2.21(s, MeCO); ESI-MS (þ): 275 [M þ Na]þ, 253
[M þ H]þ.
3.4. In vitro assays
The sensitivity of 25 phytopathogenic fungal strains and oomycetes (listed in Table 1) to
PCA and 2-OH P was assessed in an agar plate bioassay. A 5-mm-diameter plug was
excised from the margin of a 7-day-old colony of each fungal strain and placed in the
centre of a 60 mm-diameter petri plate containing PDA or PDA plates amended with three
different concentrations (10, 25 and 50 mg mLꢀ1) of PCA and 2-OH P. The four derivatives
of PCA (3, 4, 5 and 6) were evaluated against a set of 11 fungal strains at a concentration
of 25 mg mLꢀ1 under the same experimental conditions described above. Each combination
of fungal strain/amended PDA and fungal strain/non-amended PDA was duplicated in all
the trials. Once inoculated, petri plates were incubated at 21ꢁC for 6 days, and radial
growth was measured at the end of the trial. Growth inhibition of fungal isolates by 2-OH
P, PCA and its derivatives was quantified according to the following formula: [(Rc ꢀ Rs)/

964
G. Puopolo et al.
Rc] %, where Rc stands for the fungal colony radius on non-amended PDA plates while Rs
indicates fungal colony radius on amended PDA plates. The values 0% and 100%
indicated no growth inhibition and complete growth inhibition, respectively. The
experiment was replicated thrice. Results were analysed by ANOVA and the significance
of differences was compared using Tukey’s test.
3.5. Assays in liquid culture
This experiment was carried out in order to better determine the sensitivity to PCA of
those fungi that were able to overgrow on PDA plates amended with 25 mg mLꢀ1 of this
compound. Phytophthora cinnamomi, P. ultimum and R. solani were employed in this
assay. Five tubes containing 40 mL of potato dextrose broth (PDB) only and five tubes
containing PDB amended with 25 mg mLꢀ1 of PCA were inoculated with a 5-mm-diameter
plug excised from the margin of 7-day-old colony of each fungal strain. Tubes were
incubated on a rotary shaker (150 rpm) at 21ꢁC for 6 days. At the end of the incubation
period, mycelia were collected from each tube, dried under vacuum for 30 min and then
weighed. The significance of differences between the average of mycelium weight from
PDB and PCA-amended PDB was compared by Student’s t test. At the same time,
supernatants were collected in new tubes and extracted according to the procedure
previously reported in order to survey if any PCA degradation had occurred in the
presence of the growing fungi. Five uninoculated tubes containing PDB and PDB
amended with PCA at 25 mg mLꢀ1, incubated at the same conditions reported above, were
used as controls. The supernatants of strain M71 were extracted as previously reported,
and the organic extracts were dried with Na2SO4, filtered and evaporated under reduced
pressure. The extracts were resuspended in MeOH at a final concentration of 1 mg of
extracts for 1 mL. A volume of 3 mL from each sample was further analysed by TLC (SiO2,
eluent CHCl3-i-PrOH 95:5). The same volume of PCA solution (1 mg mLꢀ1) was loaded on
TLC on silica gel to check for the presence of degradation products.
4. Conclusions
Data reported in this work show that the carboxyl group of PCA is a structural feature
important for its antifungal activity, which remained substantially unaltered only in the
derivatives 3 and 4. Furthermore, this work provides useful information for the
improvement of the applications of P. chlororaphis subsp. aureofaciens strain M71,
describing the host range of plant pathogenic fungi sensitive to this compound.
Supplementary material
1H and 13C NMR and ESIMS spectra of compounds 1–6 are available online as
Figures S1–S13.
Acknowledgements
The authors wish to thank Prof. Leland S. Pierson III for kindly reviewing this manuscript. This
work was supported in part by a grant from the Italian Ministry of University and Research
(MIUR) and in part by a grant from Regione Campania- SeSIRCA ‘Recupero della fertilita dei
suoli’ Contribution DISSPAPA N. 258. A. Evidente is associated to Istituto di Chimica
Biomolecolare del CNR, Pozzuoli, Italy.

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965
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