Activity of extracts from wild edible herbs against postharvest fungal diseases of fruit and vegetables

Postharvest Biology and Technology 61 (2011) 72–82

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Activity of extracts from wild edible herbs against postharvest fungal diseases of fruit and vegetables Maria Antonia Gatto a,b , Antonio Ippolito b , Vito Linsalata a , Nicholas A. Cascarano a , Franco Nigro b , Sebastiano Vanadia a , Donato Di Venere a,∗ a b

Institute of Sciences of Food Production, National Research Council (CNR), Via G. Amendola 122/O, 70126 Bari, Italy Department of Plant Protection and Applied Microbiology, University of Bari “Aldo Moro”, Via G. Amendola 165/A, 70126 Bari, Italy

a r t i c l e

i n f o

Article history: Received 12 August 2010 Accepted 12 February 2011 Keywords: Antimicrobials Conidia germination Germ tube elongation Phenols Postharvest rot Storage

a b s t r a c t The use of plant extracts could be a useful alternative to synthetic fungicides in the management of rot fungi during postharvest handling of fruit and vegetables. The aim of this study was to assess the in vitro and in vivo activity of extracts obtained from nine wild edible herbaceous species (Borago officinalis, Orobanche crenata, Plantago coronopus, P. lanceolata, Sanguisorba minor, Silene vulgaris, Sonchus asper, Sonchus oleraceus, and Taraxacum officinale) against some important postharvest pathogens, i.e. Botrytis cinerea, Monilinia laxa, Penicillium digitatum, P. expansum, P. italicum, Aspergillus carbonarius, and A. niger. Phenolic composition of all extracts was evaluated by HPLC. Several derivatives of caffeic acid, of the flavones apigenin and luteolin, and of the flavonols kaempferol and quercetin, were identified. Extracts from S. minor and O. crenata showed the highest efficacy in all the trials. In particular, S. minor completely inhibited in vitro the conidial germination of M. laxa, P. digitatum, P. italicum, and A. niger and strongly reduced those of B. cinerea; O. crenata extract showed a lower but still significant reduction of conidial germination on all the tested fungi. Moreover, the extracts from both species were effective in reducing the germ tube elongation also when a slight inhibition of conidial germination was observed. In many cases, a dose effect was observed, with an increase of antifungal activity as the phenolic concentration increased. In trials performed on wounded fruit, S. minor extract completely inhibited brown rot on apricots and nectarines; O. crenata extract strongly reduced grey mould, brown rot, and green mould on table grapes, apricots and nectarines, and oranges, respectively. The inhibition efficacy of extracts was ascribed to the presence of some caffeic acid derivatives and/or flavonoids. HPLC phenolic analyses provided useful information to identify the possible active compounds. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Fungal infections are the main cause of postharvest rots of fresh fruit and vegetables during storage and transport, and cause significant economic losses in the commercialization phase. The most important postharvest fungal pathogens are Botrytis cinerea Pers. ex Fr., Monilinia laxa (Ehrenb) Sacc., Penicillium spp., and Aspergillus spp. (Eckert and Ogawa, 1988). The postharvest control of these pathogens is quite efficiently performed by synthetic chemical fungicides (Förster et al., 2007). However, the reduced number of authorized active ingredients, increased resistance of some postharvest fungal pathogens against the few authorized fungicides, and growing consumer demand for both high quality and safe fruit and vegetables, have increased efforts to develop alternative or complementary control means (Ippolito et al., 2005;

∗ Corresponding author. Tel.: +39 080 5929305; fax: +39 080 5929374. E-mail address: [email protected] (D. Di Venere). 0925-5214/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2011.02.005

Smilanick et al., 2008; Droby et al., 2009; Sanzani et al., 2009a; Sharma et al., 2009; Casals et al., 2010; Mari et al., 2010). Extracts obtained from many plants have recently gained popularity and scientific interest for their antibacterial and antifungal activity (Lee et al., 2007; Verástegui et al., 2008; Santas et al., 2010). Many results have been reported on the antimicrobial properties of plant extracts containing different classes of phenolic compounds (Rauha et al., 2000; Al-Zoreky, 2009). Phenolic compounds represent a rich source of biocides and preservatives that have been explored for a long time as postharvest alternative control means (Lattanzio, 2003; Schena et al., 2008). In particular, many studies have pointed out the antimicrobial efficacy of certain classes of phenolic compounds, such as hydroxybenzoic acid derivatives (Lattanzio et al., 1996; Amborabé et al., 2002; Veloz-García et al., 2010), coumaric and caffeic acid derivatives (Zhu et al., 2004; Widmer and Laurent, 2006; Korukluoglu et al., 2008), flavonoids ˜ et al., 2006; Sanzani et al., and coumarins (Ojala et al., 2000; Ortuno 2009b), catechin, epicatechin, proanthocyanidins, and tannins (Di Venere et al., 1998; Terry et al., 2004; Engels et al., 2009; Parashar

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Table 1 Wild edible herbaceous species used in the experiments. Most of them are used in traditional recipes. Scientific name

Family name

Common name

Used part

Traditional uses

Borago officinalis L. Orobanche crenata Forsk. Plantago coronopus L. Plantago lanceolata L. Sanguisorba minor Scop. s. l. Silene vulgaris (Moench) Garcke Sonchus asper (L.) Hill Sonchus oleraceus L. Taraxacum officinale Weber

Boraginaceae Orobanchaceae Plantaginaceae Plantaginaceae Rosaceae Caryophyllaceae Asteraceae Asteraceae Asteraceae

Borage Broomrape Crowfoot plantain Buckhorn plantain Salad burnet Bladder campion Spiny sow thistle Annual sow thistle Common dandelion

Leaves, flowers Stems Leaves Leaves Leaves Leaves, stems Leaves Stems, leaves, flowers Flowers, leaves

Salad, boiled, fried, soup, stuffing, vegetable pie Boiled, fried Salad, boiled, fried Salad, boiled, fried, soup Salad, boiled, grilled, soup Salad, boiled, fried, soup, vegetable pie Salad, boiled, soup Salad, boiled, soup, vegetable pie Salad, boiled, fried, soup, stuffing, vegetable pie

et al., 2009; Yoshida et al., 2009). Moreover, some authors have studied the relationship between molecular structure and antimicrobial activity of some phenolic compounds (Lattanzio et al., 1994; Amborabé et al., 2002; Bisogno et al., 2007). Among wild herbaceous species known in Italy, about 800, belonging to 91 families, are classified as edible (Conti et al., 2005). They are interesting from an ethno-botanical point of view, since a lot of them are used both in traditional recipes as raw vegetables (Bianco, 1989) and in popular medicine as a source of alternative drugs. In fact, they are known as a rich source of antioxidant, anti-inflammatory, diuretic, antibacterial, and antiviral active substances, with medicinal as well as cosmetic applications (Yukawa et al., 1996; Dhiman and Chawla, 2005; Wang et al., 2006; Di Venere et al., 2009). To our knowledge, the antimicrobial activity of phenolic extracts obtained from wild edible species against postharvest fungal pathogens has been rarely explored. Therefore, the objective of the present study was to evaluate the in vitro activity of extracts from nine wild edible plants as antimicrobial agents against major postharvest fungal pathogens. The phenolic content and composition of the extracts were evaluated. Their antifungal activity was also tested on artificially inoculated fruit, to obtain preliminary data on the efficacy in preventing disease development. 2. Materials and methods 2.1. Plant material and chemical reagents Plants of nine wild edible herbaceous species (Borago officinalis, Orobanche crenata, Plantago coronopus, P. lanceolata, Sanguisorba minor, Silene vulgaris, Sonchus asper, Sonchus oleraceus, and Taraxacum officinale) were collected from Murgia plateau (Apulia region, Southern Italy) in spring and classified according to botanical name and family (Table 1). Only the edible part was selected for trials, this being leaves for all species, except O. crenata, whose stems were used. A representative and randomized amount of fresh plant material (at least 2 kg per species) was dried in a ventilated oven at 40 ◦ C until constant weight (36–48 h), finely ground in a grinder to obtain a dry powder, and stored under vacuum in a cool room until use. The dry matter content was evaluated. High performance liquid chromatography (HPLC) grade water was obtained by a Milli-Q system (Millipore, Bedford, MA, USA). Methanol (HPLC grade) was obtained from Carlo Erba Reagents (Milan, Italy). Caffeic acid and chlorogenic acid were purchased from Sigma (Sigma–Aldrich, Milan, Italy); chicoric acid, verbascoside, and isoverbascoside were from Phytolab GmbH & Co. KG (Vestenbergsgreuth, Germany); apigenin-7-glucoside, luteolin7-glucoside, quercetin-3-glucoside, and kaempferol-3-glucoside were from Extrasynthèse (Genay, France). All HPLC standards had a chromatographic purity >95%. Potato Dextrose Agar (PDA) and Potato Dextrose Broth (PDB) were purchased from Oxoid Ltd (Berkshire, UK).

2.2. Preparation of plant extracts To prepare extracts of each species, an amount of dry powder corresponding to 50 g of fresh weight of plant tissue was extracted twice with refluxing 80% aqueous methanol (1:5, w/v) for 1 h. After extraction, the methanolic extracts were filtered through Whatman filter paper and evaporated to dryness under reduced pressure at 35 ◦ C, using a rotary evaporator. The residue was dissolved in 50 mL of potassium phosphate buffer (KH2 PO4 –K2 HPO4 , 0.1 M, pH 5.5) to give a stock solution with conventional 1× concentration (50 g of fresh matter in 50 mL of buffer). This solution was centrifuged at 10,000 × g, the supernatant filtered through 0.22 ␮m sterile pore size membrane filter (Millipore, Bedford, MA, USA), and then stored at −20 ◦ C until use. Afterwards, 0.25×, 0.5×, and 0.75× dilutions in the same buffer were prepared using the 1× stock solution. These dilutions were then used for in vitro and in vivo activity assays. 2.3. HPLC analysis of phenolic compounds The HPLC analysis of phenolic compounds in the extracts was performed using an Agilent 1100 Series liquid chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with binary gradient pump (Agilent P/N G1312A) and spectrophotometric photodiode array detector (DAD) (Agilent P/N G1328A). The Agilent ChemStation (Rev. A.06.03) software was used for spectra and data processing. An analytical Phenomenex (Torrance, California, USA) Luna C18 5 ␮m (250 mm × 4.6 mm) column at 35 ◦ C in thermostatic oven (Agilent P/N G1316A) was used for peak separation. A binary gradient elution at a flow rate of 1 mL min−1 with methanol (solvent A) and 5% (v/v) acetic acid in deionized water (solvent B) was used. The elution profile was as follow: 0–25 min = 15–40% A in B; 25–30 min = isocratic 40% A in B; 30–45 min = 40–63% A in B; 45–47 min = isocratic 63% A in B; 47–52 min = 63–100% A in B; 52–56 min = isocratic 100% A, and then back to the equilibrium conditions (15% A in B). 2.4. Identification and quantification of phenolic compounds The identification of phenolic compounds (Table 2) was performed by comparison of chromatographic retention times and UV spectra obtained by DAD with those of commercial standards, when available. The attribution of partially identified HPLC peaks as derivatives of caffeic acid or apigenin, luteolin, quercetin, and kaempferol, was performed on the basis of UV spectra, which are generally characteristic of the different classes of phenolic compounds, i.e. caffeic acid derivatives and flavonoids (Marston and Hostettmann, 2006). Caffeic acid, being absent in all extracts, was added to each sample as an internal standard and used to calculate the relative retention time of each peak in the different HPLC chromatograms (Table 2). The concentration of the identified phenolic compounds in the extracts was assessed using calibration curves made with

74 Table 2 Phenolic compound identity and content in different wild edible herbs evaluated by HPLC. Phenolic class attribution was made by UV spectra obtained from the HPLC-DAD analyses. Retention times were normalized to caffeic acid as internal standard (I.S.). Values are the mean ± SD of three replicates. Retention timea (min)

Phenolic compound identityb

Phenolic compound content (mg 100 g−1 DM)

SPECIES Borago officinalis

a b

CA – der. CA – der. CA – der. CA – der. CA – der. Chlorogenic acid CA – der. CA – der. CA – der. Caffeic acid (I.S.) CA – der. CA – der. CA – der. CA – der. A – der. A – der. Chicoric acid CA – der. A – der. CA – der. Verbascoside CA – der. CA – der. Q – der. A – der. L-7-glucoside CA – der. Isoverbascoside A – der. Q – der. Q-3-glucoside K – der. CA – der. A-7-glucoside K – der. K – der. Q – der. K – der. K-3-glucoside A – der.

Plantago coronopus 5±1

Plantago lanceolata

Sanguisorba minor

Silene vulgaris

Sonchus asper

Sonchus oleraceus

Taraxacum officinale

47 ± 5 199 ± 21 116 ± 13

261 ± 28

252 ± 27

76 ± 9 149 ± 17 85 ± 10

151 ± 19

52 ± 7 18 ± 2 20 ± 3 35 ± 4 60 ± 8 7±1 7±1

12 ± 1 12 ± 1 50 ± 6 467 ± 55

5±1

1431 ± 121 27 ± 3

18 ± 2

44 ± 5 4621 ± 221

73 ± 8 3547 ± 171

1245 ± 133

2370 ± 152

14 ± 2 3389 ± 157

5±1 55 ± 6

9±2

53 ± 6 45 ± 6 177 ± 20 200 ± 22

36 ± 4 147 ± 16

463 ± 52

362 ± 40

224 ± 24

174 ± 21

5±1 22 ± 3 73 ± 7

102 ± 12 561 ± 51 61 ± 6 228 ± 25 43 ± 6 22 ± 3 62 ± 7 98 ± 11 25 ± 3

Retention time normalized to caffeic acid as internal standard (I.S.). Phenolic derivatives (der.) of caffeic acid (CA), apigenin (A), luteolin (L), quercetin (Q), and kaempferol (K).

214 ± 22 79 ± 9

43 ± 6

M.A. Gatto et al. / Postharvest Biology and Technology 61 (2011) 72–82

4.77 5.22 5.44 5.85 6.44 7.04 7.18 9.24 9.28 9.50 9.65 10.47 10.82 11.14 11.65 14.51 14.65 15.02 15.32 16.42 17.69 18.41 18.53 19.18 19.46 19.83 19.89 21.28 21.54 22.09 22.54 23.33 23.57 24.85 24.92 26.20 27.30 27.54 27.73 34.60

Orobanche crenata

GTLcontrol



× 100

where GTL = average germ tube length of conidia. All the experiments, performed in a sterile environment, were made in triplicate and repeated twice.

538 4707 3671 3138 2528 969 3086 2202 2723

± ± ± ± ± ± ± ± ±

60 328 239 236 313 138 346 267 221

Phenolic concentration (mg L−1 )

9.3 12.5 7.5 8.4 17.0 14.0 13.0 10.0 10.0

± ± ± ± ± ± ± ± ±

0.1 0.2 0.1 0.2 0.3 0.3 0.2 0.1 0.1

Dry matter (%)

57 199 251 187 155 82 226 243 192 ± ± ± ± ± ± ± ± ± 214 ± 22

579 3765 4894 3736 1487 692 2374 2202 2723 170 ± 10

463 ± 52 362 ± 40 43 ± 6

658 ± 62

156 ± 18

K – der. Q – der.

9 82 24 21

L – der.

± ± ± ± 256 ± 29 421 ± 49 310 ± 34

79 692 224 174 28 42 30 16 62 ± ± ± ± ±

Other CA – der.

248 377 273 189 535

Phenolic derivatives (der.) of caffeic acid (CA), apigenin (A), luteolin (L), quercetin (Q), and kaempferol (K).

GTLsample

a



IGTE = 100 −

1431 ± 121 1245 ± 133 2370 ± 152

where %GC = average percentage of germinated conidia. Moreover, the length of 100 germinated conidia in each droplet was measured by using an ocular micrometric scale, and the average germ tube length was calculated both in treated samples and in buffer controls; the inhibition of germ tube elongation (IGTE) was calculated as follows:

3389 ± 157 4621 ± 221 3547 ± 171

× 100

Verbascoside

%GCcontrol

5±1



Chicoric acid

%GCsample

Borago officinalis Orobanche crenata Plantago coronopus Plantago lanceolata Sanguisorba minor Silene vulgaris Sonchus asper Sonchus oleraceus Taraxacum officinale



ICG = 100 −

A – der.

The in vitro antimicrobial activity of extracts was tested using a micro-assay method on slides. Specifically, 400 ␮L of culture medium were prepared by mixing 50 ␮L of PDB (24 g L−1 in distilled water), 50 ␮L of spore suspension, and the complementary amounts of 1× plant extract solution and distilled water required to realize final extract concentrations of 0.25×, 0.5×, and 0.75×. Phosphate buffer 0.1 M, pH 5.5, was used as a control. Afterwards, three equidistant droplets (20 ␮L each) of mixture were deposited on the surface of a slide. The slide was incubated in a Petri dish at 25 ± 1 ◦ C. After 15 h, spore germination (length/width ratio ≥2) and germ tube elongation was evaluated by observations under the microscope (40× magnification). The number of total and germinated conidia within three different microscope fields of each droplet were counted, and the average percentage of germinated conidia was calculated both in treated samples and in buffer controls; the inhibition of conidial germination (ICG) was calculated as follows:

CA derivatives

2.6. Inhibitory effect of plant extracts on in vitro pathogen growth

75

Total phenolic content (mg 100 g−1 DM)

Extracts were individually tested in vitro against seven fungal pathogens: B. cinerea, the causal agent of grey mould, M. laxa, inducing brown rot of stone fruit, P. digitatum (Pers.) Sacc., causing green mould of citrus fruit, P. italicum Wehmer, causing blue mould of citrus fruit, P. expansum Link., causing blue mould of apple, A. carbonarius (Bainier) Thom and A. niger van Tieghem, the causal agents of black mould. The pathogens were isolated from infected fruit and vegetables and cultured on PDA (39 g L−1 in distilled water) at 25 ± 1 ◦ C for 5 d. Pure cultures were maintained at 4 ◦ C in PDA tubes. Spores were harvested from 2-week-old PDA fungal cultures grown at 25 ± 1 ◦ C, by adding a few millilitres of water in each Petri dish. Suspensions were filtered through two layers of cheesecloth, and the concentration was adjusted to 104 spores mL−1 .

Content of phenolic derivatives (mg 100 g−1 DM)a

2.5. Fungal pathogens

Species

the corresponding commercial standards. Unidentified caffeic acid derivatives were quantified as caffeic acid, whereas to quantify the different flavone and flavonol derivatives, apigenin-7-glucoside, luteolin-7-glucoside, quercetin-3-glucoside, and kaempferol-3glucoside were used, each one for the derivatives of the corresponding aglycone. The content of phenolic compounds detected in each species (mean ± SD of three replicates on three different extract preparations) is reported in Table 2. The total phenolic content was calculated as the sum of the single phenolic compound content. The phenolic concentration of 1× extracts was calculated from total phenolic content, considering the dry matter percentage of each species (Table 3).

Table 3 Total phenolic content, and subdivided by class, of the plant species and phenolic concentration of extracts at 1× conventional dilution, calculated by means of dry matter (DM). Values are the mean ± SD of three replicates. The contents of chicoric acid and verbascoside, already present in Table 2 are reported for an easier comparison with the total phenolic content.

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2.7. Activity of plant extracts on artificially inoculated fruit Trials were conducted on table grapes (Vitis vinifera L.) cv Italia, orange (Citrus sinensis L.) cv Washington Navel, nectarine (Prunus persica (L.) Batsch var. laevis Grey) cv Big Bang Maillarà, and apricot (Prunus armeniaca L.) cv Reale, fruit purchased from local growers. Fruit, selected for uniform size, stage of ripening, and absence of visible defects and injuries, were surface-disinfected by dipping for 2 min in 2% sodium hypochlorite, rinsed with tap water and allowed to dry. They were treated with the plant extracts and then inoculated with spore suspensions of the pathogens: M. laxa for nectarine and apricot, P. digitatum for orange, and B. cinerea for table grapes. Nectarines, apricots, and oranges, were wounded once in the equatorial zone (2 mm wide and 2 mm deep), and 30 ␮L of extract (1× concentration) were pipetted into each wound, allowing the droplet to be absorbed into the fruit. Two hours later, wounds were inoculated with 10 ␮L of the conidial suspension (5 × 104 spores mL−1 ). Two types of control replacing the plant extracts were prepared, the first, using 0.1 M K-phosphate buffer solution pH 5.5, the second (“chemical control”), using 30 ␮L of commercial fungicide solutions containing as active ingredient “fenhexamid” for apricot and nectarines, and “imazalil” for oranges at label dosage (Droby et al., 1998). For table grapes, single berries were wounded by pulling away the peduncle, treated with the plant extracts and inoculated with the pathogen as described above. In this case, only K phosphate buffer was used as a control. Trials were arranged in a completely randomized experimental design, with 6 replicates per treatment and a different number of fruit/berries per replicate (8, 9 and 20 fruit for nectarines, apricots, and oranges, and 15 berries for table grapes, respectively). Fruit were placed in trays, packaged in plastic bags (RH = 95–98%) and stored at 15 ± 1 ◦ C (apricots and nectarines), 7 ± 1 ◦ C (oranges) and 20 ± 1 ◦ C (table grapes). All the experiments were repeated twice. Disease severity was periodically evaluated by measuring the diameter of the infected wounds or, in the case of table grapes, by using an empirical scale based on 6 values, where 0 = no rot; 1 = 1–5% rotten berry; 2 = 6–15% rotten berry; 3 = 16–30% rotten berry; 4 = 31–50% rotten berry; 5 = 51–75% rotten berry; 6 = >75% rotten berry). Moreover, the percentage of infected fruit/berries was also recorded. 2.8. Statistical analysis Data were subjected to analysis of variance (ANOVA) using Statistica Software (version 6.0; StatSoft Inc., Tulsa, OK, USA). Duncan’s Multiple Range Test (DMRT; P ≤ 0.01) and least significant difference values (LSD; P ≤ 0.01) were used to compare the means (Gomez and Gomez, 1984). Data expressed as percentages were transformed into arcsin square root values to normalise distributions before analysis of variance; however, the percentages are shown as untransformed data. The experiments were repeated twice. Data from the two experiments were combined, since statistical analysis determined homogeneity of variances according to Bartlett’s test. 3. Results 3.1. Phenolic composition and concentration of plant extracts Different classes of phenols were detected in the plant extracts. In particular, caffeic acid derivatives and flavonoids were identified by a combined analysis of HPLC chromatograms and UV spectra; other unidentified peaks were found in some cases.

Some specific compounds were identified in different amounts in more than one species. In particular, concerning caffeic acid derivatives, chlorogenic acid (in both Sonchus species), chicoric acid (in both Sonchus species, T. officinale, and B. officinalis), verbascoside and isoverbascoside (in O. crenata and both Plantago species) were found. As to flavonoids, luteolin-7-glucoside (in both Sonchus species and T. officinale), quercetin-3-glucoside (in S. minor), apigenin-7-glucoside (in both Sonchus species), and kaempferol 3-glucoside (in S. minor) were found (Table 2). With the exception of S. vulgaris, all species contained caffeic acid derivatives, which represented the main class of phenols in O. crenata, P. coronopus, P. lanceolata, and T. officinale. Derivatives of the flavones apigenin and luteolin, and of the flavonols kaempferol and quercetin, were detected in the other species (Tables 2 and 3). Within the same plant genus, i.e. the two species of Plantago and Sonchus, the same HPLC phenolic patterns were found. The phenolic pattern of T. officinale was similar to that observed in the two Sonchus species, particularly for the presence of a large amount of chicoric acid, an unidentified caffeic acid derivative (retention time 5.44 min), and a small amount of luteolin-7-glucoside; nevertheless, the apigenin-7-glucoside, present in both Sonchus species, was not found in T. officinale (Table 2). S. minor showed the richest phenolic pattern (derivatives of caffeic acid, apigenin, quercetin, kaempferol, and many other unidentified peaks), whereas in S. vulgaris only apigenin derivatives were detected. Verbascoside was found as the main phenolic compound in both O. crenata and the two Plantago species, together with isoverbascoside and another unidentified caffeic acid derivative (Table 2). Phenolic content of the plant species and phenolic concentration at 1× conventional dilution of extracts are reported in Table 3. In this table, the presence of large amounts of chicoric acid in T. officinale and Sonchus species, and verbascoside, in O. crenata and Plantago species, is pointed out. Chicoric acid content of T. officinale (2370 mg 100 g−1 DM), S. asper (1431 mg 100 g−1 DM), and S. oleraceus (1245 mg 100 g−1 DM), represented about 87%, 60%, and 57% of the total phenolic content of each species (i.e. 2723, 2374, and 2202 mg 100 g−1 DM, respectively); whereas, verbascoside content of O. crenata (3389 mg 100 g−1 DM), P. coronopus (4621 mg 100 g−1 DM), and P. lanceolata (3547 mg 100 g−1 DM), was found to be about 90%, 94%, and 95% of the total phenolic content of the corresponding species (i.e. 3765, 4894, and 3736 mg 100 g−1 DM, respectively) (Table 3). As to the remaining species, S. minor showed an intermediate total phenolic content (1487 mg 100 g−1 DM) and B. officinalis the lowest content (579 mg 100 g−1 DM); moreover, the whole S. vulgaris phenolic content (692 mg 100 g−1 DM) was attributed to apigenin derivatives (Table 3). With regard to extract concentrations, O. crenata extract showed the highest total phenolic concentration (4707 mg L−1 ), followed by P. coronopus (3671 mg L−1 ), the lowest concentrations being those found in B. officinalis (538 mg L−1 ) and S. vulgaris (744 mg L−1 ) extracts. The concentrations of the remaining extracts were between 2200 and 3100 mg L−1 (Table 3). 3.2. Inhibitory effect of plant extracts on in vitro pathogen growth The inhibitory effect of extracts was tested at three concentrations (0.25×, 0.50×, and 0.75×) on spore germination and germ tube elongation of B. cinerea, M. laxa, P. expansum, P. digitatum, P. italicum, A. carbonarius, and A. niger, however, only data at 0.75× concentration are reported (Table 4). In particular, S. minor extract showed the best results, completely inhibiting M. laxa, P. digitatum, P. italicum, and A. niger, and significantly reducing (P ≤ 0.01) conidial germination and germ tube elongation of B. cinerea and

0A 5 AB 31 D 10 ABC 25 CD 100 E 4 AB 17 BCD 14 ABC 3 AB

0A 47 B 87 DE 81 CD 81 CD 100 E 42 B 39 B 69 C 45 B

0A 10 ABC 40 E 23 DEF 21 CDE 32 DE 10 ABC 13 BCD 10 ABC 4 AB

0A 35 C 39 C 27 BC 8 AB 45 C 4A 13 AB 27 BC 0A

P. expansum (Table 4). Moreover, on most of the considered fungi, extracts from O. crenata, P. coronopus, P. lanceolata, and B. officinalis, induced an inhibition of conidial germination and/or germ tube elongation higher than 60% (P ≤ 0.01) (Table 4). Similarly, extracts from S. oleraceus and S. asper showed a significant activity against P. expansum, P. italicum, and A. niger, and against P. digitatum, respectively. A. carbonarius was the less sensitive pathogen to the plant extracts (Table 4). The control did not show inhibition of conidial germination nor germ tube elongation after incubation at 25 ± 1 ◦ C for 15 h. In general, most of the extracts were more effective in reducing the germ tube elongation than in inhibiting conidial germination. The effect of S. minor and O. crenata extracts on B. cinerea, M. laxa, and P. digitatum is shown in Fig. 1. A partial and a complete inhibition of conidia germination was observed on B. cinerea (Fig. 1b) and M. laxa (Fig. 1d), respectively, whereas hyphae malformations, disorganization of the cellular wall, and leakage of cytoplasmatic material were observed on P. digitatum (Fig. 1f). From a combined analysis of inhibition data obtained with the three utilized concentrations (0.25×, 0.50×, and 0.75×), S. minor showed complete inhibition of M. laxa, P. digitatum, P. italicum, and A. niger also at the lowest concentration used. A remarkable dose effect was observed for S. minor against B. cinerea, P. expansum, and A. carbonarius, P. lanceolata against P. digitatum and P. italicum, P. coronopus against B. cinerea, P. expansum, and A. carbonarius, and O. crenata against A. carbonarius (data not shown).

0A 35 B 49 C 7A 93 D 100 D 1A 4A 2A 2A 0A 70 C 86 C 81 C 78 C 90 C 3A 6A 73 C 0A

0A 79 DEF 87 EF 7A 90 F 100 F 49 BC 40 B 66 CDE 56 BCD

IGTE (%)

ICG, inhibition of conidial germination; IGTE, inhibition of germ tube elongation; bold type, % inhibition ≥ 60%.

0A 8 ABC 35 E 20 CD 19 D 47 E 16 CD 7 ABC 9 BCD 0A 0A 36 B 86 FG 72 EF 89 F 100 G 55 CD 67 DE 49 BC 42 BC 0A 1A 32 B 8A 76 C 100 D 5A 7A 11 A 12 A 0A 71 D 80 DE 84 DE 81 DE 100 E 17 AB 33 BC 52 C 5A 0A 22 B 56 C 24 B 30 B 100 D 10 AB 10 AB 9 AB 11 AB 0A 60 CD 75 DE 60 CD 39 BC 92 E 28 B 48 BCD 28 B 38 BC

ICG (%)

0A 47 E 60 FG 71 H 30 D 93 I 1 AB 5 ABC 11 C 9 BC Control Borago officinalis Orobanche crenata Plantago coronopus Plantago lanceolata Sanguisorba minor Silene vulgaris Sonchus asper Sonchus oleraceus Taraxacum officinale

Aspergillus carbonarius

ICG (%) IGTE (%)

Aspergillus niger

ICG (%) IGTE (%)

Penicillium italicum

ICG (%) IGTE (%)

Penicillium expansum

ICG (%) IGTE (%) ICG (%)

Monilia laxa

IGTE (%) ICG (%)

IGTE (%)

Penicillium digitatum

77

3.3. Activity of plant extracts on artificially inoculated fruit

Botrytis cinerea

Fungi Species

Table 4 Effect of plant extracts at 0.75× conventional dilution on conidia germination and germ tube elongation of the different postharvest fungal pathogens. Activity of extracts was tested using a micro-assay method on slides after 15 h of incubation in Petri dish at 25 ± 1 ◦ C. In each column values followed by the same letters are not significantly different according to DMRT at P ≤ 0.01.

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Plant extracts showed different activity in inhibiting disease development in inoculated nectarines, apricots, oranges, and table grapes. The efficacy of the different extracts in reducing disease severity and incidence of infected fruit (%) is shown in Figs. 2–5. In nectarines and apricots, brown rot due to M. laxa was completely inhibited by S. minor extract after 6 d at 15 ± 1 ◦ C (Figs. 2 and 3). Moreover, extracts of O. crenata and B. officinalis in nectarines, and O. crenata and P. coronopus in apricots showed 47% and 40% (Fig. 2a), and 75% and 57% (Fig. 3a) of lesion diameter reduction, respectively, as compared to the control (buffer-treated fruit); the corresponding incidence of infected fruit were 53% and 88% (Fig. 2b), and 44% and 71% (Fig. 3b), respectively. The extracts of S. vulgaris and T. officinale in nectarines and S. vulgaris in apricots did not affect rot development, whereas the other species showed intermediate but still significant (P ≤ 0.01) values of rot inhibition. The efficacy of S. minor extract remained unchanged after 11 d of storage at 15 ± 1 ◦ C in both nectarines and apricots. Moreover, after 24 d storage, rot development in S. minor-treated apricots was comparable to that found in the chemical control (i.e. fenhexamid-treated fruit) (data not shown). Also in oranges, S. minor extracts showed the best results in inhibiting P. digitatum green mould development after 25 d at 7 ± 1 ◦ C, the reduction being 92% as compared to the buffer-treated fruit (Fig. 4a) with 8% of infected fruit assessed at the same time (Fig. 4b). In the same conditions, imazalil completely inhibited infection development. Lower but still significant (P ≤ 0.01) reduction of lesion diameter and rot incidence were shown by B. officinalis (74% and 19%, respectively), S. oleraceus (70% and 20%, respectively), and O. crenata (60% and 20%, respectively) extracts (Fig. 4a and b). In table grapes, the extracts of all species induced a significant (P ≤ 0.01) reduction of grey mould disease severity after 6 d at 20 ± 1 ◦ C, with a reduction ranging from 53% (S. asper) to 23% (S. minor) (Fig. 5a). Similarly, the lowest percentage of infected berries was recorded for S. asper (30%), followed by S. minor (42%) and O. crenata (47%) (Fig. 5b).

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Fig. 1. Germinated conidia of B. cinerea (a, b), M. laxa (c, d), and P. digitatum (e, f) after 15 h of incubation in K phosphate buffer (a, c, e = controls) and with 0.75× conventional dilution of extracts from S. minor (b and d) and O. crenata (f). The arrows indicate some fungal hyphae disorganization of the cellular wall and leakage of cytoplasmatic material on P. digitatum. Image magnification: B. cinerea = 40×; M. laxa = 40×; P. digitatum = 100×.

4. Discussion In the present work, the in vitro antifungal activity of phenolic fractions of extracts from some wild edible herbaceous plants and its in vivo efficacy on a number of fruit were evaluated. Most of the species utilized in the experiments are commonly used in tradi-

tional recipes (Bianco, 1989). Among them, S. minor extract showed the highest activity, followed by that of O. crenata. The activity of the extracts could be attributed to phenolic compounds that are commonly reported to possess high levels of antimicrobial activity (Lattanzio et al., 1994, 1996; Bendini et al., 2006). Different classes of phenols were observed in the extracts

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Fig. 2. Activity of extracts from different wild edible herbs in reducing the development of M. laxa brown rot (a) and the incidence of infected fruit (b) on nectarines after 6 d of storage at 15 ± 1 ◦ C, 98% RH. Fruit were artificially inoculated after plant extract or buffer (control) application. Bars represent the SE of the mean. Lesion diameter (a) LSD(0.01) = 5.8; incidence (b) LSD(0.01) = 12.9.

Fig. 3. Activity of extracts from different wild edible herbs in reducing the development of M. laxa brown rot (a) and the incidence of infected fruit (b) on apricots after 6 d of storage at 15 ± 1 ◦ C, 98% RH. Fruit were artificially inoculated after plant extract or buffer (control) application. Bars represent the SE of the mean. Lesion diameter (a) LSD(0.01) = 6.9; incidence (b) LSD(0.01) = 14.3.

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utilized in this work. Phenols with antifungal activity can occur in plants as preformed compounds (phytoanticipin) or as toxic substances produced only after stimulation by different stresses (phytoalexins), as in the case of wounds, attack by pathogens, etc. (VanEtten et al., 1994). For example, in strawberry tissues the presence of some preformed antifungal compounds, such as proanthocyanidins, catechin, epicatechin, and gallic acid, proved to be responsible for resistance against grey mould at different stages of ripening (Di Venere et al., 1998; Terry et al., 2004). The group of the preformed compounds includes simple phenols, phenolic acids, flavanols, isoflavons, and dihydro-chalcons, while that of phytoalexins involves isoflavonoids, flavans, stilbenes, phenanthrene, pterocarpans, and furanocumarins (Lattanzio et al., 2001; Lattanzio, 2003). In our case, since the extracts derived from nonstressed plants, it is conceivable that phenolic components belong to the group of pre-existing phenols. In recent years, the attention of many researchers has been towards polyphenol bioavailability and dose-effects for humans, including toxic levels, with the aim to establish the “Dietary Reference Intake” values for polyphenols as guideline for their correct intake (Williamson and Holst, 2008). Indeed, the health safety of certain phenolic compounds could depend not only on their molecular structure but also on the ingested dose. Therefore, in order to explore doses most likely not toxic in potential in vivo applications, it was decided to use extracts with concentrations of active compounds comparable to those usually attained in food preparations. Thus our extracts contained the active substances present in 50 g of fresh tissue dissolved in 50 mL of buffer. This kind of approach involved the use of extracts with different phenolic concentrations. However, as our trials seem to demonstrate, it could be more important to evaluate the influence on antifungal efficacy of phenolic composition instead of phenolic concentration of extracts. The total phenolic concentration of the different extracts was not directly correlated with their antifungal efficacy. Indeed, from a

Fig. 4. Activity of extracts from different wild edible herbs in reducing the development of P. digitatum green mould (a) and the incidence of infected fruit (b) on oranges after 25 d of storage at 7 ± 1 ◦ C, 98% RH. Fruit were artificially inoculated after plant extract or buffer (control) application. Bars represent the SE of the mean. Lesion diameter (a) LSD(0.01) = 14.4; incidence (b) LSD(0.01) = 18.1.

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Fig. 5. Activity of extracts from different wild edible herbs in reducing the development of B. cinerea grey mould (a) and the incidence of infected berries (b) on table grapes after 6 d of storage at 20 ± 1 ◦ C, 98% RH. Single berries were wounded by pulling away the peduncle, then artificially inoculated after plant extract or buffer (control) application. The disease severity was estimated by an empirical scale with values from 0 (no rot) to 6 (more than 75% rotten berry). Bars represent the SE of the mean. Disease severity (a) LSD(0.01) = 0.48; incidence (b) LSD(0.01) = 13.9.

combined analysis of Tables 3 and 4, it can be observed that the most active extract (S. minor) showed an intermediate total phenolic concentration (2528 mg L−1 ), similar to that of T. officinale (2723 mg L−1 ), which did not show significant antifungal activity. In contrast, B. officinalis extract, showing the lowest phenolic concentration (538 mg L−1 ), displayed an appreciable efficacy against four of the seven investigated fungi (Table 4). The inhibition efficacy of extracts was probably due to the presence of particular phenolic compounds, showing specific activity against one or more fungi, as in the case of O. crenata and the two Plantago species. O. crenata was found to be rich in caffeic acid derivatives (Afifi et al., 1993; Di Venere et al., 2008) and in particular of verbascoside (acteoside) and, to a lesser extent, in its isomer isoverbascoside. According to Tamura and Nishibe (2002), the extracts of the two Plantago species also contained verbascoside. Based on these considerations, verbascoside could be one of the possible active compounds of O. crenata and the two Plantago species. Therefore, in addition to the well known antioxidant, anti-inflammatory, and antiviral properties, verbascoside also has antimicrobial activity, until now little documented (Avila et al., 1999; Sampaio de Andrade Lima et al., 2003; Egorov et al., 2004). S. minor extract, the most effective among the tested extracts, was found to contain both caffeic acid derivatives and flavonoids derived from apigenin, quercetin, and kaempferol. In particular, quercetin-3-glucoside and kaempferol-3-glucoside were found the most abundant flavonoids. In S. minor fresh tissues, the presence of gallic acid, ellagic acid, quercetin, kaempferol derivatives, and coumarins has been reported (Ayoub, 2003). Conceivably, the strong activity of S. minor extract against the tested fungi and diseases could be explained by a possible synergic effect of the complex phenolic pattern. In this regard, the cytotoxic and antimicrobial activities of quercetin-3-glucoside were recently reported

(Razavi et al., 2009), whereas other authors showed the antibacterial activity of quercetin-3-glucoside, kaempferol-3-glucoside, and apigenin, demonstrating their synergistic efficacy when present together in the same extract as compared to the activity of the compounds alone (Akroum et al., 2009). The presence of quercetin and kaempferol derivatives in B. officinalis, even though different from those found in S. minor, could explain the good efficacy shown by its extract against four of the seven tested fungi. Indeed, it is well known that frequently the aglycone is the active part of a flavonoidic molecule (Ciegler et al., 1971). T. officinale extract showed low efficacy against all fungi, despite its intermediate total phenolic concentration and its high content in chicoric acid. Similarly, considerable amounts of chicoric acid were also found in the two Sonchus species, in which there was also a fairly good quantity of the flavonoids luteolin-7-glucoside and apigenin-7-glucoside. These results seem to indicate the scarce antifungal activity of chicoric acid against the investigated fungi, whereas the presence of the two flavonoids might explain the slight efficacy of Sonchus extracts against some fungi. S. vulgaris contained only apigenin derivatives, and its extract was one of the least concentrated and effective. Its scarce activity may be ascribed to the poor efficacy of the apigenin derivatives present in this extract. These compounds, in particular the main peak at retention time 14.51 min, are different from the apigenin derivative found in S. minor or the apigenin-7-glucoside detected in the two Sonchus species. Generally, many extracts showed a more marked ability in inhibiting the germ tube elongation rather than the spore germination. Moreover, germinating conidia of different fungi incubated with S. minor, O. crenata, B. officinalis, P. lanceolata, and P. coronopus extracts, showed malformations of the germ tube, disorganization of the cell wall, and leakage of cytoplasmatic material from the hyphae. This behaviour suggests a progressive poisoning of the germ tube caused by some “toxic” compounds in the extract. The greater activity of the extracts on the germ tube elongation rather than on conidia germination is probably due to a different composition of the cell wall and to its need to absorb nourishing substances from outside, including also the “toxicants” occurring in the extract (Yuvamoto and Said, 2007). It can be assumed that the light lipophilic nature of some phenolic compounds contributes to their progressive accumulation in the cell membrane of the pathogens, thus altering its permeability and affecting some transport mechanisms (Kanaani and Ginsburg, 1992). The in vivo trials confirmed the strong efficacy shown in vitro by S. minor extract. It proved to be very effective in controlling both disease severity and incidence of infected fruit by M. laxa, in nectarines and apricots, and P. digitatum in oranges, similarly to the fungicide controls. The application of fenhexamid and any other fungicide during the postharvest phase of stone fruit is currently not authorized by Italian and European legislation; however, it was used as a reference control since their registrants are supporting postharvest use (Förster et al., 2007). In the trials performed on table grapes, the reduction of incidence and severity of grey mould induced by S. minor and all the other extracts was lower compared to other fruit. The kind of deep wound, pulling out the peduncle, may account for the observed poor activity. Besides S. minor, O. crenata extract was also effective in reducing incidence and severity of M. laxa, on nectarines and apricots, and P. digitatum attacks on oranges. Moreover, good results were obtained with extracts of P. coronopus on apricots and S. oleraceus and B. officinalis on oranges. In general, the observed activity of tested extracts can be considered preventive, since the fungal inoculation was made after the extract application. The efficacy of extracts on fruit was not always in accordance with the antifungal activity shown in in vitro trials as in case of B. officinalis, which was effective in vitro against M. laxa but not very effective on apricot or slightly

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effective in vitro on P. digitatum but active on oranges. Numerous factors can affect the biological activity of certain compounds when in contact with fruit tissue. Conceivably, in the complex host/antimicrobial compound/pathogen system, many biochemical processes can occur with different effects on biological activity of the antimicrobial compound. It could be hypothesized that the molecules of the applied antimicrobial compounds can undergo some structural changes (degradation, hydrolysis, polymerization, etc.) causing increase or loss of their original biological activity. Furthermore, the substances present in the extracts may act as elicitors of resistance through different mechanisms mediated by the host tissue (Ippolito et al., 2000; Lattanzio, 2003). 5. Conclusions The results obtained from both in vitro and in vivo trials indicated that the extracts had a variable degree of antifungal activity in relation to the different tested fungi. S. minor extract showed the highest antifungal activity, followed by O. crenata extract. To the best of our knowledge this is the first report on the activity of phenolic extracts from these wild edible herbs against postharvest fungal diseases of fruit and vegetables. Further studies are in progress to confirm the in vivo efficacy of the extracts on different species of stored fruit and vegetables, and to isolate, purify, and identify the most active antifungal compounds. Such compounds, if properly formulated and applied, could be used directly after partial purification or could serve as templates for synthetic analogues. A possible direct use is feasible since all the considered herbaceous species are utilizable for both field and greenhouse intensive cultivation, in order to produce biomass for active compound extraction. Their use as “lead structures” for the development of fungicides with low toxicity should be also considered, having regard of the success of the phenyl pyrroles, derived from pyrrolnitrin, a metabolite produced by Pseudomonas spp. (Nevill et al., 1988) and the strobilurins derived from a substance produced by the fungus Strobilurus tenacellus (Godwin et al., 1992). Acknowledgements The authors thank Prof. Vito V. Bianco, Department of Sciences of Plant Production, University of Bari “Aldo Moro”, for the helpful assistance in plant species collection and classification. References Afifi, M.S., Lahloub, M.F., El-Khajaat, S.A., Rüegger, H., Sticher, O., 1993. Crenatoside: a novel phenylpropanoid glucoside from Orobanche crenata. Planta Med. 59, 359–362. Akroum, S., Bendjeddou, D., Satta, D., Lalauoi, K., 2009. Antibacterial activity and acute toxicity effect of flavonoids entracte from Mentha longifolia. Am. Eurasian J. Sci. Res. 4, 93–96. Al-Zoreky, N.S., 2009. Antimicrobial activity of pomegranate (Punica granatum L.) fruit peels. Int. J. Food Microbiol. 134, 244–248. Amborabé, B.E., Fleurat-Lessard, P., Chollet, J.-F., Roblin, G., 2002. Antifungal effects of salicylic acid and other benzoic acid derivatives towards Eutypa lata: structure–activity relationship. Plant Physiol. Biochem. 40, 1051–1060. ˜ Avila, J.G., de Liverant, J.G., Martinez, A., Martinez, G., Munoz, J.L., Arciniegas, A., de Vivar, A.R., 1999. Mode of action of Buddleja cordata verbascoside against Staphilococcus aureus. J. Ethnopharmacol. 66, 75–78. Ayoub, N.A., 2003. Unique phenolic carboxylic acids from Sanguisorba minor. Phytochemistry 63, 433–436. Bendini, A., Cerretani, L., Pizzolante, L., Gallina Toschi, T., Guzzo, F., Ceoldo, S., Marconi, A.M., Andreetta, F., Levi, M., 2006. Phenol content related to antioxidant and antimicrobial activities of Passiflora spp. extracts. Eur. Food Res. Technol. 223, 102–109. Bianco, V.V., 1989. Wild Plants Utilizable as Vegetables and Condiment Herbs in Italy. Int. Symp. Hort. Germplasm cultivated and wild, 1989 Beijing (China), vol. 2. Int. Acad. Publishers, Beijing, China, pp. 55–64. Bisogno, F., Mascoti, L., Sanchez, C., Garibotto, F., Giannini, F., Kurina-Sanz, M., Enriz, R., 2007. Structure-antifungal activity relationship of cinnamic acid derivatives. J. Agric. Food Chem. 55, 10635–10640.

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