Antimicrobial properties of derivatives of the cationic tryptophan-rich hexapeptide PAF26

Short antimicrobial peptides represent an alternative to fight pathogen infections. PAF26 is a hexapeptide identified previously by a combinatorial approach against the fungus Penicillium digitatum and shows antimicrobial properties towards certain phytopathogenic fungi. In this work, PAF26 was used as lead compound and its properties were compared with two series of derivatives, obtained by either systematic alanine substitution or N-terminal amino acid addition. The alanine scan approach underlined the optimized sequence of PAF26 in terms of potency and permeation capability, and also the higher contribution of the cationic residues to these properties. The N-terminal addition of amino acids resulted in new heptapeptides with variations in their antimicrobial characteristics, and very low cytolysis to human red blood cells. Positive (Arg or Lys) and aromatic (Phe or Trp) residue addition increased broad spectrum activity of PAF26. Noteworthy, addition of selected residues had specific effects on the properties of derivatives of PAF26.     

In vitro effects of water activity, temperature and solutes on the growth rate of P. italicum Wehmer and P. digitatum Sacc

AIMS: To evaluate the effect of water activity (a(w) 0.98-0.89, adjusted with glycerol, sorbitol, glucose, or NaCl) and temperature (5-25 degrees C) on the lag phase and radial growth rate (mm day(-1)) of the important citrus spoilage fungi, such as Penicillium italicum and Penicillium digitatum grown in potato dextrose agar (PDA) medium. To select, among models based on the use of different solutes, a model fitting accurately the growth of these species in relation to a(w) and temperature. METHODS AND RESULTS: Extensive data analyses showed for both Penicillium species a highly significant effect of a(w), temperature, solutes and their interactions on radial growth rate (P < 0.0001). Radial growth rate was inhibited and the lag phase (i.e. the time required for growth) lengthened as the a(w) of the medium decreased. NaCl appeared to causes the greatest stress on growth when compared with other nonionic solutes. Penicillium italicum stopped growing at 0.96 a(w) and P. digitatum at 0.93 a(w). Under the dry conditions where growth was observed, P. italicum grew faster than P. digitatum at low temperature and P. digitatum remained more active at ambient temperature. Multiple regression analysis applied to the square roots of the growth rates observed in the presence of each solute showed that both the 'glycerol model' and the 'sorbitol model' yielded a good prediction of P. italicum growth and the 'sorbitol model' gave an accurate fit for P. digitatum growth, offering high-quality prediction within the experimental limits described. CONCLUSIONS: Mathematical models describing and predicting, as a function of a(w) and temperature, the square root of the radial growth rate of the agents responsible for blue and green decays are important tools for understanding the behaviour of these fungi under natural conditions and for predicting citrus fruit spoilage. SIGNIFICANCE AND IMPACT OF THE STUDY: Implementation of these results should contribute towards a more rational control strategy against citrus spoilage fungi.     

Activity and mode of action against fungal phytopathogens of bovine lactoferricin-derived peptides

AIM: To evaluate the activity against fungal phytopathogens of two synthetic peptides derived from the protein bovine lactoferricin: the antibacterial active core of six amino acid residues (LfcinB(20-25)) and an extension of 15 amino acids (LfcinB(17-31)). METHODS AND RESULTS: In vitro activity against fungal pathogens was determined and compared with that against model micro-organisms. Activity was demonstrated against fungi of agronomic relevance. Distinct antimicrobial properties in vitro were found for the two peptides. LfcinB(17-31) had growth inhibitory activity higher than LfcinB(20-25). However, LfcinB(17-31) was not fungicidal to quiescent conidia of Penicillium digitatum at the concentrations assayed, while LfcinB(20-25) killed conidia more efficiently. Microscopical observations showed that the mycelium of P. digitatum treated with LfcinB(17-31) developed alterations of growth, sporulation and chitin deposition, and permeation of hyphal cells. In experimental inoculations of mandarins, both peptides showed limited protective effect against the disease caused by P. digitatum. CONCLUSIONS: LfcinB(20-25) and LfcinB(17-31) peptides were shown to have antimicrobial activity against plant pathogenic filamentous fungi, with distinct properties and mode of action. SIGNIFICANCE AND IMPACT OF THE STUDY: LfcinB(20-25) and LfcinB(17-31) peptides offer novel alternatives to develop resistant plants by molecular breeding.     

Properties of a polygalacturonase-inhibiting protein isolated from 'Oroblanco' grapefruit

Polygalacturonase inhibiting protein (PGIP) was extracted from 'Oroblanco' grapefruit type (triploid pummelo-grapefruit) albedo tissue, purified and partially characterized. Extraction was carried out at 4°C with a high ionic strength extraction buffer. After dialysis and concentration by ultrafiltration the extract was chromatographed on concanavalin A-Sepharose. The PGIP activity was bound by the lectin and then eluted using 250 m M α -methyl mannopyranoside, resulting in a 17-fold purification of the PGIP and demonstrating its glycoprotein nature. The anion-exchange and size-exclusion chromatography steps that followed gave a PGIP that was 857-fold purified relative to the initial tissue extract, and having a 44 kDa molecular weight, as estimated by SDS-PAGE electrophoresis. PGIP inhibition activity was tested with endo-polygalacturonase (EC 3.2.1.15) produced by Penicillium italicum and Botrytis cinerea . The radial diffusion and reducing sugar assays showed that P. italicum and B. cinerea endo-PGs were affected by PGIP, whereas no endo-PG activity was detected in the culture filtrate of P. digitatum. In vitro tests revealed that PGIP inhibited P. italicum and B. cinerea growth. By contrast, the influence of PGIP on P. digitatum , growth was negligible, perhaps because this fungus does not produce endo-PG. Following heating for 10 min at 65°C the inhibitory activity of PGIP was reduced by 43%. PGIP activity decreased further as heating temperature increased, and was completely suppressed after heating at 100°C for 10 min.     

Identification and characterization of a hexapeptide with activity against phytopathogenic fungi that cause postharvest decay in fruits

A hexapeptide of amino acid sequence Ac-Arg-Lys-Thr-Trp-Phe-Trp-NH2 was demonstrated to have antimicrobial activity against selected phytopathogenic fungi that cause postharvest decay in fruits. The peptide synthesized with either all D- or all L-amino acids inhibited the in vitro growth of strains of Penicilium italicum, P. digitatum, and Botrytis cinerea, with MICs of 60 to 80 microM and 50% inhibitory concentration (IC50) of 30 to 40 microM. The inhibitory activity of the peptide was both sequence- and fungus-specific since (i) sequence-related peptides lacked activity (including one with five residues identical to the active sequence), (ii) other filamentous fungi (including some that belong to the genus Penicllium) were insensitive to the peptide's antifungal action, and (iii) the peptide did not inhibit the growth of several yeast and bacterial strains assayed. Experiments on P. digitatum identified conidial germination as particularly sensitive to inhibition although mycelial growth was also affected. Our findings suggest that the inhibitory effect is initially driven by the electrostatic interaction of the peptide with fungal components. The antifungal peptide retarded the blue and green mold diseases of citrus fruits and the gray mold of tomato fruits under controlled inoculation conditions, thus providing evidence for the feasibility of using very short peptides in plant protection. This and previous studies with related peptides indicate some degree of peptide amino acid sequence and structure conservation associated with the antimicrobial activity, and suggest a general sequence layout for short antifungal peptides, consisting of one or two positively charged residues combined with aromatic amino acid residues.     

枯草芽孢杆菌防治柑桔青绿霉病初报

室内平皿抑菌测定和果实处理试验初步结果表明,枯草芽孢杆菌对柑桔青绿霉具有较强的抑菌作用,能减少果实贮藏过程中病害的发生。     

Effect of water activity and temperature on germination and growth of Penicillium digitatum,P. italicum and Geotrichum candidum

AIMS: This study compares the effect of temperature (4-37 degrees C) and water activity (aw: 0.99-0.87) and their interactions on the germination rates, lag times prior to germination and mycelial growth 'in vitro' of Penicillium digitatum, P. italicum and Geotrichum candidum, the main postharvest pathogens affecting citrus fruits. METHODS AND RESULTS: Germination and growth were markedly influenced by temperature and aw. Generally, lag times were longer and germination and growth rates were slower when conditions of temperature and aw were far from optimum. All the studied species were able to germinate over a range of 4-30 degrees C at 0.995 aw, although in non-optimal conditions P. digitatum only reached 40-60% of germinated conidia. At low temperatures, P. italicum germinated and grew faster than P. digitatum and G. candidum, particularly at 0.95 aw. Penicillium italicum was also able to germinate and grow in the driest studied conditions (0.87 aw), while G. candidum did not germinate under 0.95 aw. CONCLUSIONS: Knowledge of the ecological requirements of these fungi is important in order to understand their behaviour in natural situations and to predict fungal spoilage on citrus fruits.     

Parallel evaluation of antimicrobial peptides derived from the synthetic PAF26 and the human LL37

The antimicrobial hexapeptide PAF26 was de novo designed towards phytopathogenic fungi of agricultural importance. To analyze its clinical potential, the activity of PAF26 has been determined against several microorganisms of clinical relevance including Staphylococcus, Candida, and several dermatophytes. For comparison purposes, the peptides KR20 and KI26 derived from the human cathelicidin LL37 were selected and fungal pathogens of agronomic relevance were included. PAF26 has similar antimicrobial activity in vitro compared to KR20 despite their different lengths and amino acid compositions. Moreover, neither peptide is lytic to human erythrocytes or keratinocytes. The hybrid peptide PAF26:KR20 showed better antimicrobial properties than the original peptides against most of the pathogens tested. The structural properties of PAF26:KR20 compared to related 26-amino acid peptides support the idea that the increment in toxicity correlates with positive charge and hydrophobicity. However, the degree of peptide helicity was not a predictor of antimicrobial activity.     

芦竹内生真菌F0238对植物病原菌的拮抗作用

从黄海海岸低盐药用植物芦竹(Arundo donax L．)中分离得到木霉属的内生真菌F0238,对其进行拮抗植物病原菌活性及作用机制的试验研究。结果表明F0238发酵液对Botrytis cinerea,Sclerotium rolfsii等8种植物病原菌有强的抑菌活性及抑制孢子萌发作用,且这种作用在发酵原液被稀释了160倍后仍然存在;对峙培养试验表明F0238菌对植物病原菌有很强的拮抗作用,拮抗机制主要为重寄生作用、营养竞争作用及抗生作用等。     

Development of a biological control method against postharvest diseases of citrus fruits

Candida oleophila strain O was previously selected for its high and reliable antagonistic activity against Botrytis cinerea and Penicillium expansum, two important wound pathogens on post-harvest apples. The application of these antagonistic strains on wound pathogens of Citrus was more recently undertaken. The efficacy of yeast (applied at several concentrations from 10(5) to 10(8) CFU/ml) was assessed against P. digitatum and P. italicum inoculated after one hours (at a concentration of 10(5), 106 and 10(7) spores/ml) on 'Clementine' and 'Valencia late' varieties. The protective levels were positively correlated with high concentration of antagonist and low concentration of pathogen. The antagonistic activity of this strain was also dependent on the incubation time before pathogen inoculation. The protective level increased with time between application of the antagonist and inoculation of fungal spores. Finally, the efficacy of biomass of C. oleophila strain O (produced at an industrial scale), and two different formulations of that biomass was assessed in comparison with fungicidal treatment (Thiabendazole) under semi-practical conditions against P. digitatum. This efficacy of strain O (whatever its formulation) was statistically comparable to that for TBZ at commercial dose, indicating that both formulations could be used as an alternative for conventional fungicide in postharvest treatments.     

Production and characterization of antifungal compounds produced by Lactobacillus plantarum IMAU10014

Lactobacillus plantarum IMAU10014 was isolated from koumiss that produces a broad spectrum of antifungal compounds, all of which were active against plant pathogenic fungi in an agar plate assay. Two major antifungal compounds were extracted from the cell-free supernatant broth of L. plantarum IMAU10014. 3-phenyllactic acid and Benzeneacetic acid, 2-propenyl ester were carried out by HPLC, LC-MS, GC-MS, NMR analysis. It is the first report that lactic acid bacteria produce antifungal Benzeneacetic acid, 2-propenyl ester. Of these, the antifungal products also have a broad spectrum of antifungal activity, namely against Botrytis cinerea, Glomerella cingulate, Phytophthora drechsleri Tucker, Penicillium citrinum, Penicillium digitatum and Fusarium oxysporum, which was identified by the overlay and well-diffusion assay. F. oxysporum, P. citrinum and P. drechsleri Tucker were the most sensitive among molds.     

Sensitivity of bacterial and fungal plant pathogens to the lytic peptides,MSI-99, magainin II,and cecropin B

In vitro and leaf disk assays of bacterial and fungal plant pathogens were conducted using three cationic lytic peptides, MSI-99, magainin II (MII), and cecropin B (CB). Growth of bacterial organisms was retarded or completely inhibited by low concentrations of these lytic peptides. The peptides also significantly reduced germination of fungal spores and growth of mycelia; however, higher concentrations of peptides were needed to inhibit fungal growth compared with those needed to inhibit bacteria. The relative efficacy of the peptides depended on the microorganism tested, but CB was the most inhibitory to the majority of the bacteria and fungi assayed. MSI-99, a synthetic derivative of MII with increased positive charge, showed equal or two- to fivefold higher antibacterial activity compared to MII in the in vitro assays. MSI-99 was also superior to MII against the oomycete, Phytophthora infestans but was slightly inferior to MII in assays with the true fungi, Penicillium digitatum and Alternaria solani. In the leaf disk assays, pretreating spores of Alternaria solani and Phytophthora infestans with the peptides at concentrations as low as 10 microg per ml led to significant reductions in the size of early blight lesions and prevented development of any late blight lesions on tomato leaf disks. Our results from in vitro and leaf disk assays suggest that MSI-99 can be used as a transgene to generate tomato lines with enhanced resistance to bacterial and fungal diseases of this crop.     

Production of the biocontrol agent Pantoea agglomerans strain CPA-2 using commercial products and by-products

The aim of this paper was to find the nitrogen and carbon sources that provide maximum biomass production of strain CPA-2 of the biocontrol agent Pantoea agglomerans and minimum cost of media, whilst maintaining biocontrol efficacy. To reduce the cost of media, commercial products and by-products were tested. P. agglomerans can be produced using a combination of nitrogen sources such as yeast extract (5 g l(-1)) and dry beer yeast (10 g l(-1)) with inexpensive carbohydrates such as sucrose (10 g l(-1)) and molasses (20 g l(-1)), respectively, maintaining the efficacy of the biocontrol agent against Penicillium digitatum and P. italicum on oranges. The results obtained in this study could be used to provide a reliable basis for a scale-up of this fermentation process to an industrial level.     

Chemical characterization and antifungal activity of essential oil of capitula from wild Indian Tagetes patula L.

Summary. The essential oil extracted by steam distillation from the capitula of Indian Tagetes patula, Asteraceae, was evaluated for its antifungal properties and analyzed by gas chromatography and gas chromatography–mass spectrometry. Thirty compounds were identified, representing 89.1% of the total detected. The main components were piperitone (24.74%), piperitenone (22.93%), terpinolene (7.8%), dihydro tagetone (4.91%), cis-tagetone (4.62%), limonene (4.52%), and allo-ocimene (3.66%). The oil exerted a good antifungal activity against two phytopathogenic fungi, Botrytis cinerea and Penicillium digitatum, providing complete growth inhibition at 10 μl/ml and 1.25 μl/ml, respectively. The contribution of the two main compounds, piperitone and piperitenone, to the antifungal efficacy was also evaluated and ultrastructural modifications in mycelia were observed via electron microscopy, evidencing large alterations in hyphal morphology and a multisite mechanism of action. Correspondence and reprints: Dipartimento delle Risorse Naturali e Culturali, Università di Ferrara, Corso Porta di Mare 2, 44100 Ferrara, Italy.     

The potential use of the Penicillium chrysogenum antifungal protein PAF,the designed variant PAFopt and its γ-core peptide Pγopt in plant protection

The prevention of enormous crop losses caused by pesticide-resistant fungi is a serious challenge in agriculture. Application of alternative fungicides, such as antifungal proteins and peptides, provides a promising basis to overcome this problem; however, their direct use in fields suffers limitations, such as high cost of production, low stability, narrow antifungal spectrum and toxicity on plant or mammalian cells. Recently, we demonstrated that a Penicillium chrysogenum-based expression system provides a feasible tool for economic production of P. chrysogenum antifungal protein (PAF) and a rational designed variant (PAF^opt), in which the evolutionary conserved γ-core motif was modified to increase antifungal activity. In the present study, we report for the first time that γ-core modulation influences the antifungal spectrum and efficacy of PAF against important plant pathogenic ascomycetes, and the synthetic γ-core peptide Pγ^opt, a derivative of PAF^opt, is antifungal active against these pathogens in vitro. Finally, we proved the protective potential of PAF against Botrytis cinerea infection in tomato plant leaves. The lack of any toxic effects on mammalian cells and plant seedlings, as well as the high tolerance to harsh environmental conditions and proteolytic degradation further strengthen our concept for applicability of these proteins and peptide in agriculture.     

几株农用拮抗链霉菌的初步研究

从山西蟒河自然保护区分离到5株链霉菌。琼脂移块法和发酵液扩散法试验表明,它们所产生的抗生素对棉花枯萎病菌(Fusarium oxysporum f.sp.vasinfectum)、棉花黄萎病菌(Verticillium dahliae)、棉花立枯病菌(Rhizoctonia solani)、水稻纹枯病菌(Pellicularia sasakii)、黄瓜枯萎病菌(Fusarium oxysporum f.sp.cucumerinum)等十多种植物病原真菌,对果蔬贮藏期间的病原真菌,如木霉(Trichodermasp.)、意大利青霉(Penicillium italicum)、根霉(Rhizopussp.)等都有明显的抑制作用。     

Evaluation of three essential oils as potential sources of botanical fungicides

In previous study, thirty essential oils were evaluated in vitro against two citrus pathogens namely Penicillium italicum Wehmer and Penicillium digitatum Sacc. Essential oils of Cinnamomum zeylanicum, Cinnamomum verum and Eugenia caryophyllus were selected because of their high inhibitory activities against both pathogens. The present study was undertaken to evaluate the in vivo activity of these essential oils. Fresh orange fruits were wounded and treated with different concentrations of essential oil (0.5, 1, and 5%) before being infected at the wound site with conidia suspensions of the tested pathogens. When applied at 5%, essential oils tested controlled totally the infections. Among the three essential oils tested, C. zeylanicum seems particularly interesting because of its high protection activity at 1% compare to the others. It reduced the disease incidence from 40 to 70% and the disease severity from 65 to 82%. Moreover no visible damage burn induced on the orange cuticle or skin was observed up to 5% of essential oil. These results strengthen the potential use of essential oils in postharvest disease management of citrus fruit as alternative to chemical fungicides.     

Biotransformations of 6',7'-dihydroxybergamottin and 6',7'-epoxybergamottin by the citrus-pathogenic fungi diminish cytochrome P450 3A4 inhibitory activity

Penicillium digitatum, as well as five other citrus pathogenic species, (Penicillium ulaiense Link, Geotrichum citri Link, Botrytis cinerea P. Micheli ex Pers., Lasiodiplodia theobromae (Pat.) Griffon & Maubl., and Phomopsis citri (teleomorph Diaporthe citri)) were observed to convert 6',7'-epoxybergamottin (1) into 6',7'-dihydroxybergamottin (2), bergaptol (3), and an opened lactone ring metabolite 6,7-furano-5-(6',7'-dihydroxy geranyloxy)-2-hydroxy-hydrocoumaric acid (4). Metabolism of 2 by these fungi also proceeded to 4. The structure of 4 was established by high resolution mass spectrometry and (1)H and (13)C NMR techniques. The inhibitory activity of 4 towards human intestinal cytochrome P450 3A4 (CYP3A4) was greatly decreased (IC(50) >172.0 μM) compared to 2 (IC(50)=0.81 μM).