Citrus phenylpropanoids and defence against pathogens. Part II: Gene expression and metabolite accumulation in the response of fruits to Penicillium digitatum infection

The effect of infection of Citrus sinensis (var. Navelina) fruits with Penicillium digitatum was studied at gene expression and metabolite levels. In this study, expression of genes involved in the phenylpropanoid pathway was studied in the flavedo (outer coloured part of the peel) and albedo (inner white part) in response to pathogen infection. Results of the time-course experiment showed that maximal expression of 10 out of 17 phenylpropanoid genes analysed occurred at 48 h post-inoculation, when decay symptoms started to appear, and mRNA levels either kept constant or decreased after 72 h post-inoculation. To further investigate the putative involvement of the phenylpropanoid pathway in the defence of citrus fruit, changes in the metabolic profile of both tissues infected with P. digitatum was studied by means of HPLC-PDA-FD. Metabolite accumulation levels along the time course suggest that flavanones, flavones, polymethoxylated flavones and scoparone are induced in citrus fruit in response to P. digitatum infection, although with different trends depending on the tissue.     

Comparative study of flavonoid and scoparone accumulation in different Citrus species and their susceptibility to Penicillium digitatum

Fungi of the genus Penicillium are responsible for substantial post-harvest losses in Citrus fruits. The results obtained following artificial inoculation of Citrus fruits with Penicillium digitatum showed that the degree of fungal development depended on the Citrus species. Thus, the mature fruit of Citrus paradisi were more susceptible to this fungus than the mature fruit of Citrus limon, Citrus sinensis,Citrus clementina Hort. ex Tan., and Citrus unshiu (mak) Marc. The results point to an inverse correlation between the degree of susceptibility of Citrus species to this fungus and the flavanone content – hesperidin in C. sinensis, C. clementina Hort. ex Tan. and C. unshiu (mak) Marc.; naringin in C. paradisi and the flavanone hesperidin and the flavone diosmin in C. limon. Thus, in C. sinensis,C. clementina Hort. ex Tan and C. unshiu (mak) Marc. the highest levels of the polymethoxyflavones, sinensetin, tangeretin, heptamethoxyflavone and nobiletin, were observed in the least susceptible varieties and viceversa. In the case of C. paradisi, no significant differences were detected in the polymethoxyflavone levels between varieties, while in C. limon, polymethoxyflavones were hardly detectable. The production of scoparone was observed in all the species and varieties studied after inoculation with the fungus, especially in C. limon fruits. Based on the evidence, it seems that flavanones, flavones, polymethoxyflavones (phytoanticipins) and scoparone (a phytoalexin) may well be involved in the defence mechanisms of Citrus fruits against P. digitatum. Depending on the Citrus species in question, the relative participation of one group of secondary compounds or another may vary.     

Determination of the change of flavonoid components as the defence materials of Citrus unshiu Marc. fruit peel against Penicillium digitatum by liquid chromatography coupled with tandem mass spectrometry

A healthy fruit peel of Citrus unshiu Marc. and one infected by Penicillium digitatum were analysed for flavonoids via high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) in the positive mode with selected ion monitoring (SIM). Among 16 flavonoid components characterised in C. unshiu Marc., four flavanones and nine flavones were identified for the first time. The identified compounds were quantified by HPLC–UV. To investigate the function of the flavonoids as defence materials, the flavonoid content change of the fruit peel inoculated with P. digitatum was monitored by HPLC. The flavonoid concentration in the infected fruit peel decreased initially after the infection and then gradually increased before finally progressively decreasing.     

Citrus phenylpropanoids and defence against pathogens. Part I: Metabolic profiling in elicited fruits

Penicillium spp. are among the major postharvest pathogens of citrus fruit. Induction of natural resistance in fruits constitutes one of the alternatives to chemical fungicides. Here, we investigated the involvement of the phenylpropanoid pathway in the induction of resistance in Navelate oranges by examining changes in the metabolic profile of upon eliciting citrus fruits. By using both HPLC-PDA-FD and HPLC-PDA-QTOF–MS allowed the identification of several compounds that seem to be relevant for induced resistance. In elicited fruits, a greater diversity of phenolic compounds was observed in the flavedo (outer coloured part of the peel) when compared to the albedo (inner white part). Moreover, only small changes were detected in the most abundant citrus flavonoids. The coumarin scoparone was among the compounds with the highest induction upon elicitation. Two other highly induced compounds were identified as citrusnin A and drupanin aldehyde. All three compounds are known to exert antimicrobial activity. Our results suggest that phenylpropanoids and their derivatives play an important role in the induction of resistance in citrus fruit.     

Inhibition of Penicillium expansum by an oxidative treatment

Several oxidizing compounds such as sodium hypochlorite (NaClO) and hydrogen peroxide (H₂O₂) are used to control postharvest decay in fresh fruit due to their antimicrobial effects. Here, we applied these compounds in vitro, in the presence of CuSO₄, against Penicillium expansum, causal agent of apple blue mold. MICs were 50 mg L⁻¹ and 400 mmol L⁻¹ for NaClO and H₂O₂, respectively, when these compounds were individually applied to conidia suspensions during 2 min. A combined oxidative treatment (OT) consisting on an incubation with 1 mg L⁻¹ NaClO and 200 mmol L⁻¹ H₂O₂, in the presence of 6 mmol L⁻¹ CuSO₄, inhibited growth, conidial germination and fungal infectivity on apple. The fractional inhibitory concentration index for the interaction between NaClO and H₂O₂ in the OT was 0.52 indicating a synergistic effect of the oxidizing compounds. These results suggest that the OT could be an interesting alternative for apple diseases postharvest control.     

The methoxyflavones in Citrus reticulata Blanco cv. ponkan and their antiproliferative activity against cancer cells

The major polymethoxyflavones in the fruit (ponkan) peels of Citrus reticulata Blanco cv. ponkan were identified as isosinensetin, sinensetin, nobiletin and tetramethyl-o-scutellarein by a combined separation using high-speed countercurrent chromatography and preparative high performance liquid chromatography, and structure elucidation by electrospray ionisation mass spectrometry (ESI-MS) and ¹H and ¹³C nuclear magnetic resonance (NMR). The antiproliferative activity of the four compounds against four cancer cell lines (A549, HL-60, MCF-7 and HO8910) showed that isosinensetin had a lower IC₅₀ value for MCF-7 and HO8910 cancer cell lines. Determination of polymethoxyflavones in ponkan peels from different cultivation regions displayed relatively steady contents of the four compounds and a higher content of isosinensetin, which suggested that ponkan peels are excellent sources of functional polymethoxyflavones that may help prevent female cancers, such as ovarian cancer and breast cancer.     

Bioassay-guided isolation and identification of antifungal components from propolis against Penicillium italicum

The present study was aimed at identification of antifungal components against Penicillium italicum from Chinese propolis with bioassay-guided fractionation technique. Propolis ethanolic extract (PEE) was separated and purified by liquid–liquid extraction and thin layer chromatography (TLC) and the most active band was subjected to HPLC–MS/MS to identify the antifungal compounds. The results showed PEE and its fractions had strong antifungal activity against P. italicum. Among the fractions of PEE partitioned by petroleum ether, ethyl acetate, n-butanol and water, ethyl acetate fraction (E-Fr) exhibited the most effective activity against P. italicum. Further bioautographic TLC assay showed Band I, with Rf value of 0.70, had an inhibitive zone, which showed the strongest antifungal activity and completely inhibited the growth of P.italicum at 200 mg/L. Bioactive components found in Band I were further identified as pinobanksin, pinocembrine, chrysin and galangin. This study exhibited Chinese propolis and its main flavonoids was potential natural alternatives for the control of citrus blue mould caused by P.italicum.     

Biotransformation of imperatorin by Penicillium janthinellum. Anti-osteoporosis activities of its metabolites

Imperatorin (IMP) is a major constituent of many herbal medicines and possesses anti-osteoporosis activity. The present research work aimed to study the biotransformation processes of IMP and evaluated the anti-osteoporosis activity of the transformed metabolites. Among 18 strains of filamentous fungi screened, Penicillium janthinellum AS 3.510 exhibited good capability to metabolise IMP to the new derivatives. Ten transformed products were isolated and purified, and their structures were identified accurately based on spectroscopic data. Eight metabolites (2–8 and 10) were novel and previously unreported. The major biotransformation reactions involved hydroxylation of the prenyloxy side-chain and the lactone ring-opening reaction of furocoumarin skeleton. In addition, anti-osteoporosis activities of all products (1–10) were evaluated using MC3T3-E1 cells. The results showed that products 5 and 8 had the best bioactivities in increasing MC3T3-E1 cell growth. These products could be used in future therapeutic regimens for treating osteoporosis.     

Chemical composition and biological activity of Citrus jambhiri Lush

The fresh peel of Citrus jambhiri was extracted with aqueous methanol and the residue was fractionated using light petroleum, chloroform and ethyl acetate. The constituents of the extracts were separated by column chromatography employing solvents of different polarity. The chemical structure of the isolated compounds was then identified by MS and NMR. Column chromatography of the petroleum fraction resulted in the isolation of nobiletin, 5-O-demethylnobiletin, tangeretin, 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone, 3,5,6,7,8,3′,4′-heptamethoxyflavone, and a mixture of β-sitosterol and stigmasterol. The chloroform fraction afforded 6-demethoxynobiletin, 5,4′-dihydroxy-6,7,8,3′-tetramethoxyflavone, limonin and nomilin. The flavonoid glycosides naringin, hesperidin and neohesperidin were isolated from the ethyl acetate fraction. The chemical structure of the isolated compounds was established by MS and NMR (APT, COSY, HSQC, HMBC, and NOESY). LC–ESI-MS analysis of the ethyl acetate fraction afforded eight flavonoid glycosides, while the dichloromethane fraction of the defatted seeds contained seven limonoid aglycones. The chloroform fraction exerted the strongest DPPH^∗ free radical scavenging activity in comparison to other fractions. The petroleum fraction showed a significant inhibition of lipoxygenase indicating an anti-inflammatory action (IC₅₀ 29 ± 1 μg/mL). Some of the isolated polymethoxyflavones exhibited strong cytotoxicity against COS7, HeLa and Caco-2 cell lines.     

The flavonoid composition of flavedo and juice from the pummelo cultivar (Citrus grandis (L.) Osbeck) and the grapefruit cultivar (Citrus paradisi) from China

The flavonoid composition of flavedos and juices from four Chinese pummelo cultivars (Citrus grandis ‘Shatianyu’, C. grandis ‘Guanximiyu’, C. grandis ‘Yuhuanyu’, and C. grandis ‘Cuixiangtianyu’) and one grapefruit cultivar (Citrus paradisi ‘Changshanhuyu’), was investigated using high-performance liquid chromatography diode array detection electrospray ionisation mass spectrometry (HPLC-DAD-ESI MS/MS). Twenty-eight flavonoids were identified. Naringin was the predominant flavanone in these pummelo varieties, whereas naringin and neohesperidin were the principal flavanones found in C. paradisi ‘Changshanhuyu’. The four pummelo cultivars had higher levels of flavones when compared with the grapefruit. Based on the flavonoid profiles in flavedos and juices, four pummelo cultivars and one grapefruit cultivar were well separated.     

The effects of the combination of Pichia membranefaciens and BTH on controlling of blue mould decay caused by Penicillium expansum in peach fruit

The feasibility of 0.2 g l⁻¹ benzo-thiadiazole-7-carbothioic acid S-methyl ester (BTH) to improve the efficacy of Pichia membranefaciens in controlling postharvest blue mould decay in peach fruit was investigated. Our results showed that biocontrol activity of P. membranefaciens against blue mould caused by Penicillium expansum in peach fruit could be enhanced by addition of 0.2 g l⁻¹ BTH. The combination of P. membranefaciens and BTH resulted in a more effective control of blue mould than individual treatment of P. membranefaciens or BTH alone. The combined treatment had a synergistic effect on the induction of superoxide dismutase, catalase, ascorbate peroxidase, chitinase and β-1,3-glucanase activities, which induced stronger disease resistance in fruit than BTH or yeast alone, and resulted in a lower lesion diameter and disease incidence of blue mould decay in peaches. Furthermore, the combined treatment did not impair the quality parameters including fruit firmness and contents of total soluble solids, titratable acidity and vitamin C of peach fruit after 6 days of storage at 20 °C. These results suggested that the use of BTH may be an effective method to improve the biological activity of P. membranefaciens.     

Antioxidative and anti-inflammatory properties of Citrus sulcata extracts

Citrus sulcata was subjected to ultrasound, high-pressure, and Soxhlet extractions. The antioxidant and anti-inflammatory activities of the extracts were evaluated qualitatively and quantitatively. The antioxidant content of the peel extract was twice that of the fruit extract. The quantitative analysis showed that the narirutin and hesperidin contents in the peel extracts were 8.8 and 7.5 mg/100 g, respectively. These extracts had a total phenolic content of 112.22 ± 2.89 gallic acid equivalent (GAE) mg/100 g, a total flavonoid content of 54.09 ± 1.01 rutin equivalent (RE) mg/100 g, DPPH radical scavenging activity of 46%, and antioxidant activity of 213.25 ± 2.82 μM of Trolox equivalents (TEAC). C. sulcata extracts could prevent hydrogen peroxide (H₂O₂)-induced oxidative stress, reduce expression of the inflammatory markers nuclear Factor kappa B (NFκB) and phosphorylated IκBα (p-IκBα) in A549 human lung carcinoma cells, and inhibit Lipopolysaccharide (LPS)-induced THP-1 monocyte differentiation to an extent of 85%.     

Conidial production by Penicillium nalgiovense for use as starter cultures in dry fermented sausages by solid state fermentation

Penicillium nalgiovense is the typical species used as starter culture on the casing of dry fermented sausages and it has been often produced by solid state fermentation (SSF). Soy beans, maize kernels and wheat bran (WB) at 50% humidity were tested as substrates in SSF for conidial production and viability in P. nalgiovense. Among them WB was the best substrate for conidial production and viability. Thus, WB proved to be a suitable and convenient choice for conidial production by P. nalgiovense in SSF. By analysing conidial production on different membranes in malt extract agar (MEA) and WB no differences were observed between different treatments for the same substrate. Therefore, the practical choice of the inert support seems to be filter paper. A natural substrate like WB together with membranes as support helped the production of viable conidia by SSF as starters for dry fermented sausages.     

Study of natural mango juice spoilage and microbial contamination with Penicillium expansum by high resolution H NMR spectroscopy

High resolution ¹H NMR has been applied to monitor the changes in the composition of natural mango juice subjected to spoilage and to microbial contamination with Penicillium expansum. A vast number of compounds undergoing changes upon these processes have been identified and their variations followed throughout time (132 h). Besides the formation of typical fermentation products (e.g. acetate, lactic acid, acetoin and isopropanol/2,3-butanediol) and the utilization of the major sugars (sucrose, glucose and fructose), there were changes in organic acids (e.g. decreases of quinic and shikimic acids with formation of 3,4,5-trihydroxycyclohexane acid in spoiled juice, and decreases of citric and malic acids in contaminated juice), amino acids (decreases of alanine, leucine, isoleucine and valine), and less abundant components such as oligosaccharides and aromatic compounds. The possibility of using these changes as early indicators of natural spoilage or P. expansum contamination is discussed.     

Odour gradients and patterns in volatile emission of different plant parts and developing fruits of grapefruit (Citrus paradisi L.)

The volatiles spontaneously emitted in vivo by different plant parts of grapefruit (Citrus paradisi Macf., Rutaceae) were collected by solid phase micro-extraction (SPME) during the whole vegetative cycle of the plant and characterised by GC–MS to verify their involvement in entomophilous pollination, a controversial topic in the biology of this species. Furthermore, the essential oils obtained by expression of the pericarp from unripe and ripe fruits were studied. Altogether 127 compounds were identified, accounting from 82.1% to 99.9% of the whole volatiles. The main constituents detected were mono- and sesquiterpenes, with limonene (0.5–95.2%), linalool (0.2–52.5%), sabinene (0.5–42.5%), myrcene (0.2–15.4%), and β-caryophyllene (0.3–41.0%) as the most represented ones.     

Inhibitory effect of sour orange (Citrus aurantium) juice on Salmonella Typhimurium and Listeria monocytogenes

The sour orange (Citrus aurantium) juice is commonly used as flavoring and acidifying agent for vegetable salads and appetizers in Turkey. It was aimed to determine the survival and growth pattern of Salmonella Typhimurium and Listeria monocytogenes in sour orange juice. Different concentrations of neutralized and un-neutralized juice samples were inoculated with each of the test microorganisms (∼6 log CFU/mL) separately and then incubated at 4 °C and 37 °C for seven days. It was detected both of the test microorganisms could survive and even grow in neutralized juice samples at 37 °C for two days. However, none of them could survive at the end of seventh day of incubation at 37 °C. Low incubation temperature (+4 °C) increased the survival of the tested microorganisms. Also, it was detected that L. monocytogenes were less resistant to the variable conditions than S. Typhimurium. It was concluded that the antimicrobial effect of sour orange juice mainly depends on the low pH value of the product. However, incubation time and temperature are also effective on the survival of the tested pathogens.     

Composition of peel essential oils from four selected Tunisian Citrus species: Evidence for the genotypic influence

The peel essential oils from four selected Tunisian Citrus species: sweet orange (Citrus sinensis Osbeck), mandarin (Citrus reticulata Blanco); sour orange (Citrus aurantium L.) and pummelo (Citrus grandis Osbeck), cultivated under the same pedoclimatic and cultural conditions have been analysed by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS). The essential oils content ranged from 1.06% to 4.62% (w/w) in pummelo and mandarin, respectively. The qualitative and quantitative analysis led to the identification of 70 components in all oil samples. The analysed oils consist mainly in monoterpene hydrocarbons (97.59–99.3%), with limonene (92.52–97.3%) and β-pinene (1.37–1.82) being the major constituents. The remaining chemical classes were weakly represented (<1%). Both qualitative and quantitative differences between oil samples have been observed and numerous components have been proposed as marker compounds. Since the influence of different environmental factors has been eliminated, the observed chemical variability between the studied species and cultivars seems likely to results from the genetic variability.     

Juice of New citrus hybrids (Citrus clementina Hort. ex Tan.×C. sinensis L. Osbeck) as a source of natural antioxidants

Fruit juices of pigmented and non-pigmented new citrus hybrids obtained by crossing clementine cv. Oroval with different cultivars of blood oranges were analysed to determine parameters related to fruit quality (total soluble solids titratable acidity, pH), and the content of ascorbic acid, total phenolics, flavanones, anthocyanins and phenolic acids. Antioxidant properties were evaluated by oxygen radical absorbance capacity (ORAC), and inhibition of induced linoleic acid peroxidation (InLAP) assays. The results of this study show that some hybrids with high antioxidant activity owing to considerable polyphenol content may be considered rich sources of phytochemicals. The OTA 9 hybrid was shown to be richest in polyphenols, suggesting that consumption of OTA 9 fruit or juice could be useful in health promotion and a disease-preventing diet. Moreover, the juice of this hybrid could be used as raw material to produce antioxidant ingredients for dietary, pharmaceutical, or cosmetic purposes.     

Evaluation of Penicillium expansum isolates for aggressiveness, growth and patulin accumulation in usual and less common fruit hosts

Experiments were carried out in vivo and in vitro with four isolates of Penicillium expansum (I 1, E 11, C 28 and I 12) to evaluate their aggressiveness, growth and patulin accumulation in both usual (pears and apples) and less common hosts (apricots, peaches, strawberries and kiwifruits) of the pathogen. The 75% of isolates showed the ability to cause blue mould in all tested hosts. In particular, C 28 and I 1 were the most and the least aggressive isolates, respectively (52.9 and 10.6% infection and 20.7 and 15.4 mm lesion diameters). ‘Candonga’ strawberries and ‘Pinkcot’ apricots showed the largest lesion diameters (29.8 and 25.3 mm), followed by ‘Conference’ pears, ‘Spring Crest’ peaches and ‘Abate Fetel’ pears. With the exception of ‘Candonga’ strawberries, the formation of colonies and mycelial growth of P. expansum isolates on fruit puree agar media (PAMs) was stimulated in comparison to a standard growth medium (malt extract agar, MEA). Two of the most aggressive isolates in our assays (I 12 and C 28) showed the greatest accumulation of patulin both in vitro and in vivo, while the least aggressive isolate (I 1) produced patulin only in a few growth media and cvs. Patulin concentration on fruit PAMs was higher than patulin detected in infected fruit tissues. Apple PAMs were the more favorable substrates for patulin accumulation in vitro (maximum concentration 173.1 and 74.1 μg/mL in ‘Pink Lady and ‘Golden Delicious’ PAMs, respectively) and ‘Pink Lady’ apples inoculated with the isolate E 11 showed the greatest accumulation of patulin in the whole in vivo assay (33.9 μg/mL). However, infected tissue of cv Golden Delicious showed lower average accumulation of patulin (1.7 μg/mL) than that of cv Pink Lady (19.1 μg/mL), and no significant differences in patulin concentrations were found among ‘Golden Delicious’ apples and tested cvs of pears, kiwifruits and strawberries. Peaches were highly susceptible to patulin accumulation, showing average concentrations of 27.4 and 18.6 μg/mL in vitro and in vivo, respectively. Apricots were also consistently positive for patulin accumulation, both in vitro (average values of 20.1 μg/mL) and in vivo (average values of 9.4 μg/mL). Our study showed the potential of some less common hosts of P. expansum (in particular peaches and apricots) to support patulin production, indicating that a steady monitoring of patulin contamination should be carried out in fruit substrates other than apples and pears.     

Antifungal activity and chemical composition of Citrus reticulata Blanco essential oil against phytopathogens from North East India

The essential oil (EO) isolated by hydro-distillation from the peel of fully matured ripen fruits of Citrus reticulata Blanco were analyzed by GC and GC-MS. Thirty seven different components were identified constituting approximately ≥99% of the oil. The major components were limonene (46.7%), geranial (19.0%), neral (14.5%), geranyl acetate (3.9%), geraniol (3.5%), β-caryophyllene (2.6%), nerol (2.3%), neryl acetate (1.1%) etc. The antifungal activity of the oil was tested by poisoned food (PF) technique and the volatile activity (VA) assay against five plant pathogenic fungi viz Alternaria alternata (Aa), Rhizoctonia solani (Rs), Curvularia lunata (Cl), Fusarium oxysporum (Fo) and Helminthosporium oryzae (Ho). The oil showed better activity in VA assay. The Minimum inhibitory concentration (MIC) for Aa, Rs and Cl was 0.2 ml/100 ml whereas >0.2 ml/100 ml for Fo and Ho in PF technique. Fungal sporulation was also completely inhibited at 2 ml/100 ml of the oil except for Cl and Ho, which was only 0.5% (±0.5) and 0.25% (±0.25) respectively as compared to control.