Potential for Development of Tolerance by Penicillium digitatum and Penicillium italicum after Repeated Exposure to Potassium Sorbate

Two strains of Penicillium digitatum and one strain of Penicillium italicum were exposed to various levels of sorbic acid and potassium sorbate, and the MICs were determined. Selected strains of the molds were then repeatedly exposed to subinhibitory levels of the compounds to determine whether increased tolerance might develop. The MIC of sorbic acid (pH 4.75) to P. digitatum was between 0.02 and 0.025%. The MIC of sorbate (pH 5.5) to two strains of P. digitatum and P. italicum was found to be between 0.06 and 0.08%. Increasing levels of sorbate resulted in increasing growth suppression of the molds. Populations of P. digitatum were tested for increased tolerance to sorbic acid, and none was found. Individual molds that started from the same parent colony were examined for increased tolerance to potassium sorbate. Two P. digitatum strains developed no observable increased tolerance, but P. italicum developed a slight increase in tolerance to sorbate. When spores of P. italicum and P. digitatum were exposed to higher levels of sorbate for prolonged times, the fungicidal or fungistatic activity of the inhibitor was dependent upon pH, length of exposure time, level of sorbate, and the mold strain.     

Evaluation of Aspergillus aculeatus GC-09 for the biological control of citrus blue mold caused by Penicillium italicum

Blue mold caused by Penicillium italicum is a severe postharvest disease in citrus fruits. In this study, the fermentation product (FP-E) of Aspergillus aculeatus GC-09, an endophytic fungus isolated from a citrus plant, was found to exhibit antifungal activity against P. italicum with a MIC of 0.3125 mg/mL. The fungus A. aculeatus GC-09 was identified based on the studies of morphology and ITS nucleotide sequence. FP-E significantly inhibited the spore germination and mycelial growth of P. italicum. Scanning electron microscopy (SEM) results of P. italicum treated with FP-E showed shrunken, distorted and collapsed hyphae and conidiospores, indicative of the cell membrane damage, which was further confirmed by the propidium iodide (PI) fluorescent staining analysis. Consistent with the microscopy observation, FP-E led to the leakage of cellular constituents from P. italicum, which is evident from the increase in electrical conductivity and nucleic acid contents in the mycelial solution incubated with FP-E. In addition, FP-E treatment considerably increased the intracellular reactive oxygen species (ROS) content, and reduced the enzyme activities of both catalase (CAT) and peroxidase (POD) in P. italicum cells. Furthermore, orange fruits treated with FP-E showed fewer disease symptoms compared to the untreated fruits. These results suggested that the antifungal activity of FP-E might be associated with the disruption of cell membrane integrity, the accumulation of ROS level, and the reduction of the antioxidant enzymes activity of P. italicum. Therefore, A. aculeatus GC-09 might be a potential microbial resource for the biocontrol of citrus postharvest blue mold.     

Inhibition of penicillium digitatum and penicillium italicum in vitro and in planta with Panomycocin, a novel exo-β-1,3-glucanase isolated from Pichia anomala NCYC 434

Panomycocin, a novel exo-beta 1,3 glucanase, was tested as an antifungal agent against green and blue mold diseases, the most important causes of post harvest decay in citrus fruits. All tested isolates of Penicillium digitatum and Penicillium italicum were susceptible to panomycocin in vitro. Effective panomycocin concentrations for 50% growth inhibition (MIC-2) for P. digitatum and P. italicum were 2 and 1 μg ml⁻¹, respectively. Complete (MIC-0) growth inhibition of all isolates observed at a panomycocin concentration of 16 μg ml⁻¹. Treatment of spores with panomycocin at values lower than the MIC-0 led to slower germ tube elongation and mycelium growth. In tests on fruit, panomycocin at concentrations equal to in vitro MIC-0 value protected lemon fruit from decay.     

Purification and characterization of antifungal compounds from Bacillus coagulans TQ33 isolated from skimmed milk powder

Bacillus coagulans TQ33, isolated from skimmed milk powder, displays strong antifungal activity against plant pathogenic fungi. The antifungal compound of the B. coagulans TQ33 culture was extracted by thin-layer chromatography and column chromatography, and its structure was elucidated based on HPLC, LC-MS, and NMR analysie. The antifungal compound was identified as phenyllactic acid (PLA), and it was found to have a minimum inhibitory concentration on Phytophthora drechsleri Tucker of 18 mg/mL. Bio-control activity tests indicated that PLA has a wide spectrum of antagonistic effects against Fusarium oxysporum, Botrytis cinerea, Glomerella cingulata, Penicillium citrinum, Penicillium digitatum, particularly against F. oxysporum. PLA is the most notable antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi that has been isolated and identified to date. These results indicate that B. coagulans TQ33 has the potential for application in biological pesticides.     

In vitro antifungal efficacy of <Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 9642 chitosan-AgNPs composite against post-harvest disease of citrus fruits

Green and blue mold postharvest diseases are the most vital negative components influencing the local market of citrus fruits. Citrus fruits were collected, and fungi were isolated. Among the fungal isolates identified, Penicillium digitatum and Penicillium italicum recorded the highest occurrence of 39.5 and 25.6%, respectively. In this work, we extracted chitosan from Aspergillum niger ATCC 9642. Fourier transform infrared spectroscopy was utilized to confirm the functional groups of the obtained compound, which exhibited the main characteristic bands of O–H stretching at 3302 cm^-1, and C–O–C band at 1125 cm^-1. A. niger ATCC 9642 chitosan had the degree of deacetylation of 88.5%, a molecular weight of 1.8 × 10⁵ Da, and viscosity of 7.3 centipoises; these values were comparable to those for standard shrimp chitosan. Ultraviolet-visible light spectra revealed the presence of A. niger ATCC 9642 chitosan-AgNPs composite. Using antifungal and spore germination assays, it was found that this composite exhibited effective antifungal action against P. digitatum and P. italicum compared with a chitosan standard. In a comet assay, the percentage of tail DNA was considered as a parameter that indicated DNA damage. The comet parameter increased significantly (P < 0.05) with A. niger ATCC 9642 chitosan–AgNPs composite, and the increase was dose-dependent. The increase in the DNA damage positively correlated with the inhibition performance of the A. niger ATCC 9642 chitosan–AgNPs composite.     

Biocontrol of green and blue molds in postharvest satsuma mandarin using Bacillus amyloliquefaciens JBC36

In order to control postharvest rot of satsuma mandarin (mandarin) fruits, a new strain of Bacillus amyloliquefaciens JBC36 (JBC36) was isolated from rhizosphere of a mandarin orchard and tested for its suppression of decay due to green and blue molds caused by Penicillium digitatum and Penicillium italicum, respectively, and its mode of action was investigated. In addition, carnauba wax-based and paraffin oil-based coating formulations were developed to increase the control efficacy of the antagonist. The strain JBC36 at 10⁸ CFU mL^?1 inhibited incidence of green and blue molds on wounded mandarin fruits with control efficacies of 88 and 80.2%, respectively. Mycelial growth and spore germination of P. digitatum and P. italicum were strongly inhibited in the presence of JBC36 or antagonistic metabolites. In order to determine antifungal activity, three kinds of antibiotics were isolated by reverse-phase high performance liquid chromatography (RP-HPLC) and identified as lipopeptide families, iturin A, fengycin and surfactin by RP-HPLC and thin-layer chromatography (TLC) analysis. Fengycin was further identified as C16 fengycin A by liquid chromatography/electrospray ionisation-mass spectrometry (LC/ESI-MS) and mass spectrometry/mass spectrometry (MS/MS) analysis. Volatile organic compounds from the antagonist also reduced the mycelial growth of P. digitatum and P. italicum. Carnauba wax-based and paraffin oil-based coating formulations containing 10⁸ CFU mL^?1 of JBC36 efficiently decreased the incidence of green mold with control efficacy of 91 and 80.9%, respectively. Overall, the antagonistic rhizobacterium JBC36 is a promising biocontrol agent for use in preventing postharvest spoilage of mandarin fruits by green and blue molds.     

Effect of bongkrekic acid on growth and metabolism of filamentous fungi

The antifungal activity of bongkrekic acid against 17 tested molds was determined. Bongkrekic acid prevented spore germination and mycelial proliferation of Aspergillus niger, Rhizopus oryzae and Penicillium italicum. The action of bongkrekic acid was fungicidal. Under these conditions, the incorporation of ¹⁴C-leucine and ¹⁴C-uracil into the perchloric acid insoluble material of germinating A. niger conidia was significantly reduced by bongkrekic acid. Respiratory activity of resting spores was not affected by bongkrekic acid. Respiratory activity of germinated spores was inhibited by bongkrekic acid to the extent of 30 to 60% of controls for A. niger, R. oryzae and P. italicum. It has been concluded that operation of adenine nucleotide translocation in mitochondria of tested fungi is obligatory both for normal spore germination and fungal growth.     

Potential of a new strain of Bacillus subtilis CPA-8 to control the major postharvest diseases of fruit

Biocontrol potential of Bacillus subtilis strain CPA-8 was tested against the main postharvest diseases of orange, apple and stone fruit. Previously, CPA-8 growth was characterized and its antifungal activity in vitro determined against Botrytis cinerea, Monilinia laxa, M. fructicola, Penicillium digitatum, P. expansum, and P. italicum. In vivo activity against these pathogens was tested by treating fruits with cells, endospores or cell free supernatants. CPA-8 treatments cannot control decay caused by P. digitatum and P. italicum on oranges. The higher concentrations of CPA-8 studied were effective in controlling B. cinerea on apple, showing grey mold incidence from 70 to 12.5% in comparison with 100% in the control. However, in general, CPA-8 treatments were not effective in controlling P. expansum. The best results of CPA-8 treatments were obtained in stone fruit against M. laxa and M. fructicola where most treatments resulted in brown rot incidence of 0% compared with 70 and 90% in the control. Based on these results, cultures, cells and cell free supernatants at different concentrations were tested against M. laxa and M. fructicola on stone fruit. Most bacterial concentrations were effective in controlling M. laxa and M. fructicola as well as or better than Serenade® Max, in some treatments showing brown rot incidences of 0% in comparison with 100% of control. Bacterial populations of CPA-8 were maintained stable or increased up to 2-log inside wounds, showing the ability of the bacteria to colonize injured tissues. Experimental evidence suggests that B. subtilis CPA-8 has biocontrol potential for control of postharvest disease on several fruit types.     

Antifungal activity of alkanols: inhibition of growth of spoilage yeasts

Primary aliphatic alkanols from C₆ to C₁₃ were tested for their antifungal activity against Saccharomyces cerevisiae using a broth dilution method. Undecanol (C₁₁) was found to be the most potent fungicide against this yeast with the minimum fungicidal concentration (MFC) of 25 μg/ml (0.14 mM), followed by decanol (C₁₀) with the minimum inhibitory concentration (MIC) of 50 μg/ml (0.31 mM). The time-kill curve study showed that undecanol was fungicidal against S. cerevisiae at any growth stages. This fungicidal activity was not influenced by pH values. Dodecanol (C₁₂) was the most effective fungistatic but did not show any fungicidal activity up to 1600 μg/mL. Fungistatic dodecanol quickly reduced cell viability, but the cell viability recovered shortly after and then finally became no longer different from the control indicating that the effect of dodecanol on S. cerevisiae was classified as a sublethal damage. However, fungistatic dodecanol combined with sublethal amount of anethole showed a fungicidal activity against this yeast. Anethole completely restricted the recovery of cell viability. Therefore expression of the synergistic effect was probably due to the blockade of the recovering process from dodecanol induced-stress. The alkanols tested inhibited glucose-induced acidification by inhibiting the plasma membrane H⁺-ATPase. Octanol (C₈) increased plasma membrane fluidity in the spheroplast cells of S. cerevisiae. The same series of aliphatic primary alkanols was also tested against a food spoilage fungus Zygosaccharomyces bailii and compared with their effects against S. cerevisiae. Decanol was found to be the most potent fungicide against Z. bailii with an MFC of 50 μg/ml (0.31 mM), whereas undecanol was found to be the most potent fungistatic with an MIC of 25 μg/ml (0.14 mM). The time-kill curve study showed that decanol was fungicidal against Z. bailii at any growth stage. This antifungal activity was slightly enhanced in combination with anethole. The primary antifungal action of medium-chain (C₉–C₁₂) alkanols comes from their ability as nonionic surfactants to disrupt the native membrane-associated function of the integral proteins. Hence, the antifungal activity of alkanols is mediated by biophysical process, and the maximum activity can be obtained when balance between hydrophilic and hydrophobic portions becomes the most appropriate.     

Sulla penetrazione di Penicillium digitatum Sacc. e Penicillium italicum Wehmer nei frutti degli agrumi

Summary

The A., with a series of controls and investigations, accomplished on different groups of Oranges and following various forms of artificial infection with Penicillium digitatum and Penicillium italicum, renders evident the different reactions that these moulds have with regard to the resistance of the cuticle and the epidermic and under-epidermic stratums of the peel of the controlled fruits. Moreover the A. confirms that both the P. digitatum and the P. italicum can act as «parasite of injury» and as «parasite of contact».     

Study of the mechanism of ε-poly-l-lysine as an antifungal on Candida albicans and Saccharomyces cerevisiae

The antimicrobial activity of ε-poly-l-lysine (EPL) has been documented, but its antifungal activity on yeast is not well defined and its mechanism of action has been vaguely explained. Our studies revealed that on both, Candida albicans and Saccharomyces cerevisiae, the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) were 250 μg·mL^?1; EPL produced a K⁺ and Ca²⁺ efflux, and at higher concentrations also an efflux of material absorbing at 260 nm, small peptides, and phosphate is produced, along with the inhibition of fermentation and extracellular acidification and respiration. Moreover, growth was inhibited, reactive oxygen species (ROS) production increased, and cell viability decreased. The polycation also produced plasma membrane potential hyperpolarization. The effects were dependent both on the cell quantity and polycation concentration, as well as the media used. The plasma membrane disruption was confirmed by TEM and PI staining.     

Pectin Lyase Production by a Penicillium italicum Strain

Growth and concomitant production of an extracellular pectin lyase (PL) [poly(methoxylgalactosiduronate) endolyase; EC 4.2.2.10] were investigated in a group of 16 fungi grown in liquid medium containing pectin as a supplementary carbon source. Culture filtrates of both Penicillium italicum (CECT 2294) and P. expansum (CECT 2275) showed the highest PL activity and contained polygalacturonase but not pectinesterase activity. The effect of the inoculum size, the carbon source (sucrose and glucose syrup), and the presence of pectin on the production of PL by P. italicum was studied. The presence of 2.6 mM glycerophosphate in the culture medium enhanced the appearance of PL but was not inhibitory for the in vitro activity. However, glycerol inhibited the enzyme nearly 50% at such a concentration.     

Antifungal Activity of Baicalein Against Candida krusei Does Not Involve Apoptosis

The present study was designed to evaluate the antifungal activity of baicalein against Candida krusei isolates. Using a broth microdilution assay, baicalein exhibited potent in vitro antifungal activity against C. krusei isolates with a minimum inhibitory concentration of 2.7 μg/ml. Flow cytometric study indicated that baicalein depolarized mitochondrial membrane potential in a concentration-dependent manner. However, mechanistic analyses showed that the intracellular reactive oxygen species (ROS) level was virtually unchanged, and massive DNA fragmentation was not observed in C. krusei isolates after baicalein treatment even at a concentration which was apoptotic in C. albicans. Taken together, we conclude that the antifungal activity of baicalein in C. krusei isolates occurs through perturbation in mitochondrial homeostasis without causing elevation of the intracellular ROS level and does not involve apoptosis.     

Effect of a novel antifungal peptide P852 on cell morphology and membrane permeability of Fusarium oxysporum

P852, a novel cyclic peptide isolated from Bacillus amyloliquefaciens L-H15, showed potent antifungal activity against several major plant fungal pathogens including Fusarium oxysporum. To elucidate the antifungal mechanism, the impact of P852 on the cell morphology and membrane permeabilization of F. oxysporum was studied. By applying electron microscopy and fluorescent techniques, we showed that P852 treatment caused the morphological change of F. oxysporum cells and disrupted its cell structure, including formation of blebs, broken hyphae, deformation of membrane, intracellular organization disruption, pore formation, and cell lysis. Our findings provide insights into the mode of action of P852, which laying a foundation to develop P852 as a novel antifungal agent to control plant fungal pathogens.     

Broad-spectrum antifungal-producing lactic acid bacteria and their application in fruit models

A large-scale screen of some 7,000 presumptive lactic acid bacteria (LAB), isolated from animal, human, or plant origin, identified 1,149 isolates with inhibitory activity against the food-spoilage mould Penicillium expansum. In excess of 500 LAB isolates were subsequently identified to produce a broad spectrum of activity against P. expansum, Penicillium digitatum, Penicillium notatum, Penicillium roqueforti, Rhizopus stolonifer, Fusarium culmorum, Aspergillus fumigatus and Rhodotorula mucilaginosa. Partial 16S rRNA sequencing of 94 broad spectrum isolates revealed that the majority of antifungal producers were strains of Lactobacillus plantarum. The remaining population was composed of Weissella confusa and Pediococcus pentosaceous isolates. Characterization of six selected broad-spectrum antifungal LAB isolates revealed that antifungal activity is maximal at a temperature of 30 °C, a pH of 4.0 and is stable across a variety of salt concentrations. The antifungal compound(s) was shown to be neither proteinaceous nor volatile in nature. P. pentosaceous 54 was shown to have protective properties against P. expansum spoilage when applied in pear, plum and grape models, therefore representing an excellent candidate for food-related applications.     

Effect of some essential oils on mycelial growth of <Emphasis Type="Italic">Penicillium digitatum</Emphasis> Sacc.

The antifungal action of four essential oils of Foeniculum vulgare (fennel), Thymus vulgaris (thyme), Eugenia caryophyllata (Clove) and Salvia officinalis (sage) was tested in vitro against Penicillium digitatum Sacc. Direct contact and vapour phase were used to test the antifungal activity of these essential oils against P. digitatum that is responsible for green mould rot of citrus fruits. The vapour phase and direct contact of clove and thyme essential oils exhibited the strongest toxicity and totally inhibited the mycelial growth of the test fungus. Thyme and clove essential oils completely inhibited P. digitatum growth either when added into the medium 600 μl l⁻¹ or by their volatiles with 24 μl per 8 cm diameter Petri dish. In in vitro mycelial growth assay showed fungistatic and fungicidal activity by clove and thyme essential oils. Sage and fennel oils did not show any inhibitory activity on this fungus. Scanning electron microscopy (SEM) was done to study the mode of action of clove oil in P. digitatum and it was observed that treatment with the oil leads to large alterations in hyphal morphology.     

Accumulation of Scoparone in Heat-Treated Lemon Fruit Inoculated with Penicillium digitatum Sacc

Phytoalexin scoparone (6,7-dimethoxycoumarin) generally was not detected in noninoculated lemon fruit (Citrus limon [L.] Burm., cv Eureka) but accumulated in fruit after inoculation with Penicillium digitatum Sacc. A much greater increase in the amount of scoparone was found in fruit exhibiting an incompatible response to Penicillium after heat treatment at 36°C for 3 days. Heat treatment prevented development of decay in the inoculated fruit. The concentration of the compound after inoculation continued to increase during and after the heat treatment period, reaching 178 micrograms per gram fresh weight of the flavedo 6 days after the heat treatment. Changes in scoparone concentration in fruit were closely correlated with the changes in the antifungal activity of the fruit extract. A low concentration of the phytoalexin was detected in fruit injured mechanically. Scoparone also accumulated in the fruit following ultraviolet illumination; the concentration of the compound was dose-dependent. Median effective dose values of the inhibition of germ tube elongation and spore germination of P. digitatum were 29 and 46 micrograms per milliliter, respectively. Our findings suggest that the rapid increase in scoparone concentration plays an important role in the increased resistance of heat-treated lemon fruit to infection by P. digitatum.     

Ergosterol depletion under bifonazole treatment induces cell membrane damage and triggers a ROS-mediated mitochondrial apoptosis in Penicillium expansum

Penicillium expansum is the causal agent of blue mold in harvested fruits and vegetables during storage and distribution, causing serious economic loss. In this study we seek the action modes of bifonazole against this pathogen. Bifonazole exhibited strong antifungal activity against P. expansum by inhibiting ergosterol synthesis. The ergosterol depletion caused damage to the cell structure and especially cell membrane integrity as observed by SEM and TEM. With increased unsaturated fatty acids contents, the cell membrane viscosity decreases and can no longer effectively maintain the cytoplasm, which ultimately decreases extracellular conductivity, changes intracellular pH and ion homeostasis. Exposure of hyphal cells to bifonazole shows that mitochondrial respiration is inhibited and reactive oxygen species (ROS) levels–including H₂O₂ and malondialdehyde (MDA) – are significantly increased. The functional impairment of mitochondria and cell membrane eventually cause cell death through intrinsic apoptosis and necroptosis.     

Silver nanoparticle production by the fungus Fusarium oxysporum: nanoparticle characterisation and analysis of antifungal activity against pathogenic yeasts

The microbial synthesis of nanoparticles is a green chemistry approach that combines nanotechnology and microbial biotechnology. The aim of this study was to obtain silver nanoparticles (SNPs) using aqueous extract from the filamentous fungus Fusarium oxysporum as an alternative to chemical procedures and to evaluate its antifungal activity. SNPs production increased in a concentration-dependent way up to 1 mM silver nitrate until 30 days of reaction. Monodispersed and spherical SNPs were predominantly produced. After 60 days, it was possible to observe degenerated SNPs with in additional needle morphology. The SNPs showed a high antifungal activity against Candida and Cryptococcus , with minimum inhibitory concentration values ≤ 1.68 µg/mL for both genera. Morphological alterations of Cryptococcus neoformans treated with SNPs were observed such as disruption of the cell wall and cytoplasmic membrane and lost of the cytoplasm content. This work revealed that SNPs can be easily produced by F. oxysporum aqueous extracts and may be a feasible, low-cost, environmentally friendly method for generating stable and uniformly sized SNPs. Finally, we have demonstrated that these SNPs are active against pathogenic fungi, such as Candida and Cryptococcus .     

Antifungal activity of extracts from Atacama Desert fungi against Paracoccidioides brasiliensis and identification of Aspergillus felis as a promising source of natural bioactive compounds

Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values≤ 125.0 µg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 µg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 µg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.