Fungitoxicity of the essential oil of Citrus sinensis on post-harvest pathogens

Summary The essential oil extracted from the epicarp of Citrus sinensis exhibited absolute fungitoxicity against the 10 post-harvest pathogens. GC–MS studies of the oil revealed the presence of 10 chemical constituents, of which limonene was found to be the major component (84.2%). The activity of the oil was tested by the poisoned food technique (PF) and the volatile activity (VA) assay and the oils showed greater toxicity in the VA assay than in the poisoned food assay. The nature of the toxicity was studied in the VA assay and it was observed that the oil was fungicidal for the 10 pathogens in the 700 ppm (mg/l) to 1000 ppm range. The oil was extremely toxic for spore germination and it was found that at 700 ppm, spore germination was inhibited in the 10 test fungi out of the 12 tested. Treatment at 300 ppm concentration exhibited 70–100% inhibition of spore germination in most of the fungi tested. Scanning electron microscopy (SEM) was done to study the mode of action of the oil in Aspergillus niger and it was observed that treatment with the oil leads to distortion and thinning of the hyphal wall and the reduction in hyphal diameter and absence of conidiophores.     

Keratinolysis and its morphological expression in hair digestion by airborne fungi

The morphological expression of keratinolysis in fungi isolated from the air of Torino (98 isolates belonging to 36 species) was studied. Light microscopy on whole material and on semithin sections, as well as scanning electron microscopy was used. There were 19 keratinolytically active species, with seven in the genusChrysosporium (C. indicum, C. keratinophilum, C. pannicola, C. tropicum, C. an.Arthroderma cuniculi, C. an.Pectinotrichum llanense, C. an.Renispora flavissima), four in the genusMalbranchea (M. arcuata, M. fulva, M. sulphurea, M. st.Uncinocarpus reesii), and three in the genusTrichophyton (T. mentagrophytes, T. rubrum, T. terrestre). In addition there wereAphanoascus fulvescens, Beauveria bassiana, Geomyces pannorum v.pannorum, Gymnoascus umbrinus andMyceliophthora vellerea. Most of these species were capable of developing structures related to surface erosion and radial penetration contemporaneously. HoweverGymnoascus umbrinus, Myceliophthora vellerea, an isolate ofC. indicum, C. tropicum andTrichophyton mentagrophytes demonstrated only surface erosion. Different isolates of one species can vary in their production of invasive structures and in degree of keratinolytic activity. Thus such activity, like many biochemical activities of fungi, does not appear to be a constant or rigorously species-specific character.     

Biodegradation of ivory (natural apatite): possible involvement of fungal activity in biodeterioration of the Lewis Chessmen

Fungal biodeterioration of ivory was investigated with in vitro inoculation of samples obtained from boar and walrus tusks with the fungi Aspergillus niger and Serpula himantioides, species of known geoactive abilities. A combination of light and scanning electron microscopy together with associated analytical techniques was used to characterize fungal interactions with the ivory, including changes in ivory composition, dissolution and tunnelling, and the formation of new biominerals. The research was aimed at providing further understanding of the potential roles of fungi in the colonization and deterioration of ivory in terrestrial environments, but also contributes to our knowledge regarding the possible origins of the surface damage observed on early medieval sculptures made largely from walrus tusks, referred to as ‘the Lewis hoard of gaming pieces’, that were presumably produced for playing chess. The experiments have shown that the possibility of damage to ivory being caused by fungi is realistic. Scanning electron microscopy revealed penetration of fungal hyphae within cracks in the walrus tusk that showed also widespread tunnelling by fungal hyphae as well as ‘fungal footprints’ where the surface was etched as a consequence of mycelial colonization. Similar phenomena were observed with boar tusk ivory, while production of metabolites could lead to complete dissolution of the sample. Colonization of ivory and/or exposure to fungal activity lead to extensive secondary biomineral formation, and this was identified as calcium oxalate, mainly as the monohydrate, whewellite.     

Antifungal activity and mode of action of silver nano-particles on Candida albicans

In this study, the antifungal effects of silver nano-particles (nano-Ag) and their mode of action were investigated. Nano-Ag showed antifungal effects on fungi tested with low hemolytic effects against human erythrocytes. To elucidate the antifungal mode of action of nano-Ag, flow cytometry analysis, a glucose-release test, transmission electron microscopy (TEM) and the change in membrane dynamics using 1,6-diphenyl-1,3,5-hexatriene (DPH), as a plasma membrane probe, were performed with Candida albicans. The results suggest nano-Ag may exert an antifungal activity by disrupting the structure of the cell membrane and inhibiting the normal budding process due to the destruction of the membrane integrity. The present study indicates nano-Ag has considerable antifungal activity, deserving further investigation for clinical applications. K.-J. Kim and W. S. Sung contributed equally to this work and should be considered co-first authors.     

Effect of chitosan on hyphal growth and spore germination of plant pathogenic and biocontrol fungi

Aims: To investigate the toxic effect of chitosan on important root pathogenic and biocontrol fungi (nematophagous, entomopathogenic and mycoparasitic). Methods and Results: We have used standard bioassays to investigate the effect of chitosan on colony growth and developed bioassays to test spore germination. The results showed that the root pathogenic and mycoparasitic fungi tested were more sensitive to chitosan than nematophagous and entomopathogenic fungi. Chitosanases (and perhaps related enzymes) are involved in the resistance to chitosan. Two fungi, one sensitive to chitosan, Fusarium oxysporum f. sp. radicis‐lycopersici, and one less sensitive, Pochonia chlamydosporia, were selected for ultrastructural investigations. Transmission electron microscopy revealed differences in the ultrastructural alterations caused by chitosan in the spores of the plant pathogenic fungus and in those of the nematophagous fungus. Confocal laser microscopy showed that Rhodamine‐labelled chitosan enters rapidly into conidia of both fungi, in an energy‐dependent process. Conclusions: Nematophagous and entomopathogenic fungi are rather resistant to the toxic effect of chitosan. Resistance of nematophagous and entomopathogenic fungi to chitosan could be associated with their high extracellular chitosanolytic activity. Furthermore, ultrastructural damage is much more severe in the chitosan sensitive fungus. Significance and impact of the study: The results of this paper suggest that biocontrol fungi tested could be combined with chitosan for biological control of plant pathogens and pests.     

Colonization ofRetama raetam seeds by fungi and their significance in seed germination

Examination by scanning electron microscopy and incubation on potato-dextrose agar medium showed that dry seeds ofRetama raetam were externally free of fungi. When planted in sandy loam soil, the seeds become colonized with eleven soil-borne fungal species. The fungi were isolated on cellulose agar, pectin agar and lignin agar media.Aspergillus flavus, A. niger, A. fumigatus, Penicillium capsolatum andFusarium oxysporum had broad occurrence and were recovered on all the three media. The production of hydrolytic enzymes by the isolated fungi depends on the substrate and species.Penicillium capsolatum, P. spinulosum andA. niger had wide enzymatic amplitude and they were able to produce cellulolytic, pectolytic and lignolytic activities on corresponding substrates as well as on seed-coat-containing media. The lignolytic activities of the isolated species exceptChaetomium bostrychodes andTrichoderma viride were enhanced by applying the seed-coat materials as C- source rather than lignin. SoakingR. raetam seeds in culture filtrates of most of the fungi grown on seed-coat-supplemented media induced a pronounced and distinct stimulating effect on seed germination. The most effective filtrates were those ofP. capsolatum, P. spinulosum andSporotrichum pulverulentum.     

Antifungal activity of essential oils and their synergy with fluconazole against drug-resistant strains of Aspergillus fumigatus and Trichophyton rubrum

The aim of this study was to screen certain plant essential oils and active compounds for antifungal activity and their in vitro interaction with fluconazole against drug-resistant pathogenic fungi. The methods employed in this work included disc diffusion, broth macrodilution, time kill methods and checkerboard microtiter tests. Oil compositions were evaluated by gas chromatography-mass spectrometry (GC-MS) analysis. Transmission electron microscopy was used to assess the effect of essential oils on cellular structures of test fungi. Test fungal strains exhibited resistance to at least two drugs (fluconazole and itraconazole). Among the 21 essential oils or active compounds tested, ten showed promising antifungal activity. GC-MS analysis revealed the presence of major active compounds in the essential oils used. Cinnamaldehyde showed the most promising antifungal activity and killing potency against Aspergillus fumigatus MTCC2550 and Trichophyton rubrum IOA-9. Cinnamaldehyde showed strongest synergy with fluconazole against A. fumigatus and T. rubrum by reducing the minimum inhibitory concentration of fluconazole up to 8-fold. Zones of lysis of the cell wall and cell membrane appeared to be where cinnamaldehyde acted on fungi. This study highlights the broad spectrum antifungal activity of essential oils and active compounds and their synergy with fluconazole against drug-resistant fungi.     

鰤诺卡氏菌对大口黑鲈头肾巨噬细胞的侵染过程

【背景】鱼类诺卡氏菌病潜伏期和病程较长,感染率和死亡率较高,给水产养殖业带来较大的经济损失,其病原鰤诺卡氏菌(Nocardia seriolae)是胞内寄生菌,侵入细胞后引起慢性感染是主要的致病机制。【目的】构建鰤诺卡氏菌侵染大口黑鲈(Micropterus salmoides)头肾巨噬细胞体外模型,观察鰤诺卡氏菌侵染巨噬细胞的过程并探究鰤诺卡氏菌对巨噬细胞的凋亡作用。【方法】采用密度梯度离心法分离巨噬细胞,通过特异性染色和PCR扩增巨噬细胞表达基因mpeg1对细胞进行鉴定,并通过CCK-8法和氧呼吸暴发活性测定检测巨噬细胞的活性;通过倒置荧光显微镜和流式细胞术观察侵染过程中细菌与细胞的形态与数量变化;通过双荧光流式细胞术检测、乳酸脱氢酶(lactate dehydrogenase, LDH)释放试验及线粒体膜电位检测,探究鰤诺卡氏菌对巨噬细胞的凋亡作用。【结果】从大口黑鲈头肾分离获得纯度高的巨噬细胞,经染色和PCR法鉴定为巨噬细胞;筛选出最优的体外培养条件为1640培养基+1%青霉素链霉素+1%胎牛血清。在脂多糖刺激后,巨噬细胞的氧呼吸暴发能力显著提高(P<0.05)。GFP-鰤诺卡氏菌侵染细胞2 h后细菌被细胞吞噬,4 h细胞变圆且贴壁率降低,6 h细菌大量繁殖并包围细胞,8 h后细胞大量死亡。凋亡相关实验结果表明,侵染初期巨噬细胞凋亡率增加,LDH释放增加,线粒体膜电位下降;随着侵染时间延长,细胞凋亡率下降、LDH释放量及线粒体膜电位下降减少,说明鰤诺卡氏菌对巨噬细胞起先促进后抑制凋亡的作用。【结论】通过密度梯度离心法成功分离大口黑鲈头肾巨噬细胞,并通过鰤诺卡氏菌侵染细胞后初步摸清鰤诺卡细菌在细胞水平的致病机理,建立了鰤诺卡氏菌侵染大口黑鲈头肾巨噬细胞的体外模型;证实了鰤诺卡氏菌可侵染巨噬细胞并抑制细胞凋亡,从而达到在巨噬细胞内存活,为进一步开展鰤诺卡氏菌与巨噬细胞相互作用并阐明鰤诺卡氏菌的致病机制奠定了研究基础。     

Isolation of endophytic actinomycetes from selected plants and their antifungal activity

The isolation of endophytic actinomycetes from surface-sterilized tissues of 36 plant species was made using humic acid–vitamin (HV) agar as a selection medium. Of the 330 isolates recovered, 212 were from roots, 97 from leaves and 21 isolates from stems with a prevalence of 3.9, 1.7 and 0.3%, respectively. Identification of endophytic actinomycetes was based on their morphology and the amino acid composition of the whole-cell extract. Most isolates were classified as Streptomyces sp. (n = 277); with the remainder belonging to Microbispora sp. (n = 14), Nocardia sp. (n = 8) and Micromonospora sp. (n = 4). Four isolates were unclassified and 23 were lost during subculture. The most prevalent group of isolates were the Streptomyces sp. occurring in 6.4% of the tissue samples of Zingiber officinale. Scanning electron microscopy investigation of this plant revealed that 7.5% of the root and 5% of the leaf samples contained endophytes. Three of the Streptomyces sp. isolates strongly inhibited Colletotrichum musae, five were very active against Fusarium oxysporum and two strongly inhibited growth of both test fungi.     

Potential of Cactoblastis cactorum as a Vector for Fungi Pathogenic to Pricklypear, Opuntia inermis

In Australia, fungi associated with larvae of the biological control agent Cactoblastis cactorum may contribute to the control of the exotic weed pricklypear (Opuntia inermis). C. cactorum larvae were assessed for their ability to vector pathogenic fungi into O. inermis by the infestation of larvae with fungal suspensions. Six fungal isolates caused disease after being carried into the host on external surfaces of larvae, and propagules of one isolate (UQ5109) initiated disease after being transferred from the cladode epidermis into the host by larvae feeding on the plant. Scanning electron microscopy revealed extensive hyphal growth on the external surfaces of larvae infested with several of the isolates. Fungi isolated from field-grown O. inermis cladodes were tested for pathogenicity to this plant in an in vivo plant assay. In total, 152 isolates were screened, 22 of which infected the host in pathogenicity tests. Only 1 (UQ5115) infected undamaged host tissue, whereas the remainder required the host to be wounded before infection could proceed. The majority of isolates were only weakly pathogenic, even when inoculated via wounds, suggesting that most were either saprophytes or weak parasites. This study demonstrates that it is possible for larvae of C. cactorum to transmit fungal pathogens into O. inermis tissue and it has provided a sound basis for future field work to determine the contribution that fungi make to the control of O. inermis.     

Morphological and phagocytic characteristics of peritoneal exudate cells in tilapia, Oreochromis niloticus (Trewavas), and carp, Cyprinus carpio L.

The morphology and phagocytic activity of peritoneal exudate cells (PEC) obtained by an intraperitoneal injection of liquid paraffin into tilapia, Oreochromis niloticus , and carp, Cyprinus carpio , were studied with light and electron microscopy. PEC consisted of monocyte-macrophage series cells (M-Mø), neutrophils, eosinophils (granular cells) and others. Cells exhibiting the same morphology as mammalian macrophages but different from monocytes of the same species were identified with light and electron microscopy and designated as peritoneal macrophages. Light and electron microscopy revealed that M-Mø, neutrophils and eosinophils (granular cells) phagocytozed foreign materials added in vivo and in vitro. Eosinophils appeared later in the peritoneal exudate and less actively phagocytic as compared with M-Mø and neutrophils. Small and large phagosomes were formed in M-Mø, neutrophils and eosinophils (granular cells). Large phagosomes were common in neutrophils. Fusion of cytoplasmic granules with the phagosome membrane was observed. The in vitro experiment on phagocytosis revealed that the phagocytic rates in M-Mø and neutrophils were positively correlated with the doses of foreign materials. The results indicated that these two cell types have the highest capacity of phagocytosis.     

The Interaction of Naegleria fowleri Amoebae with Murine Macrophage Cell Lines

The present study was undertaken to determine whether murine macrophage cell lines exhibited in vitro amoebicidal activity comparable to that elicited by activated murine peritoneal macrophages. Peritoneal macrophages activated in vivo by bacillus Calmette-Guérin or Propionibacterium acnes demonstrated significant cytolysis of Naegleria fowleri amoebae. The macrophage cell line RAW264.7 also effected cytolysis of amoebae, but to a lesser extent than that elicited by activated peritoneal macrophages. However, the macrophage cell lines, J774A.1 and P388D₁, did not exhibit amoebicidal activity. Macrophage conditioned medium prepared from RAW264.7 macrophages mediated cytolysis of L929 tumor cells but had no effect on N. fowleri amoebae. In addition, neither recombinant tumor necrosis factor nor recombinant interleukin-1 exhibited amoebicidal activity. Scanning electron microscopy of co-cultures revealed that N. fowler bound to activated peritoneal macrophages and RAW264.7 macrophages. These results suggest that RAW264.7 macrophages treated in vitro with lipopolysaccharide are similar to macrophages activated in vivo in that they effect contact-dependent cytolysis of Naegleria fowleri amoebae. The RAW264.7 macrophages are unlike primary macrophage cultures in that they either do not release soluble amoebicidal factors into the conditioned medium or they release insufficient quantities.     

Cellular interactions between arbuscular mycorrhizal fungi and rhizosphere bacteria

Summary We have investigated whether direct physical interactions occur between arbuscular mycorrhizal (AM) fungi and plant growth promoting rhizobacteria (PGPRs), some of which are used as biocontrol agents. Attachment of rhizobia and pseudomonads to the spores and fungal mycelium ofGigaspora margarita has been assessed in vitro and visualized by a combination of electron and confocal microscopy. The results showed that both rhizobia and pseudomonads adhere to spores and hyphae of AM fungi germinated under sterile conditions, although the degree of attachment depended upon the strain.Pseudomonas fluorescens strain WCS 365 andRhizobium leguminosarum strains B556 and 3841 were the most effective colonizers. Extracellular material of bacterial origin containing cellulose produced around the attached bacteria may mediate fungal/bacterial interactions. These results suggest that antagonistic and synergistic interactions between AM fungi and rhizosphere bacteria may be mediated by soluble factors or physical contact. They also support the view that AM fungi are a vehicle for the colonization of plant roots by soil rhizobacteria.Abbreviations AM arbuscular mycorrhiza - PGPR plant growth promoting rhizobacteria - CBH cellobiohydrolase - DAPG 2,4-(diacetyl-phloroglucinol - TY triptone-yeast - LB Lauria-Bertani Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Fungi in sediments of the sea of Japan and their biologically active metabolites

The most abundant marine fungi encountered in various regions of the Sea of Japan belong to the generaPenicillium, Aspergillus, Wardomyces, Trichoderma, Chrysosporium, andChaetomium. Facultative marine fungi of the generaScytalidium, Verticillium, andOidiodendron and obligate marine fungi of the genusDendryphiella are much less abundant. The composition of marine sediments and the anthropogenic load on them were found to influence the abundance and species diversity of fungi, as well as the occurrence of fungal strains producing hemolytically active substances. The biodiversity of mycobiota and the abundance of hemotoxin-producing fungi in marine sediments may be used to evaluate the anthropogenic load on marine biocenoses. Hemolytic compounds were produced by 57% of the fungi isolated from marine sediments. The hemolytic activity ofChaetomium spiculipilium was revealed in the fraction of the culture liquid containing extracellular fatty acids and pigments. The fatty acid composition of this marine fungus was determined.     

Chicory extracts from Cichorium intybus L. as potential antifungals

In this work extracts from roots of the common vegetable Cichorium intybus L., highly appreciated for its bitter taste, were studied to investigate their possible biological activity on fungi from a variety of ecological environments: some are parasites on plants (phytopathogens) or of animals and humans (zoophilic and anthropophilic dermatophytes), others live on the soil and only seldom parasitize animals (geophilic dermatophytes). The extracts were ineffective on geophilic species and on tested phytopathogens, with the exception of Pythium ultimum, whereas they inhibited the growth of zoophilic and anthropophilic dermatophytes, in particular Trichophyton tonsurans var. sulfureum, whose treatment caused morphological anomalies, here observed by scanning electron microscopy. This behaviour is discussed on the basis of the presence in the chicory extract of the two main sesquiterpene lactones, 8-deoxylactucin and 11β,13-dihydrolactucin.     

Effect of chitosan on physiological, morphological, and ultrastructural characteristics of wood-degrading fungi

An investigation was undertaken to understand the mechanism(s) by which chitosan exerts its antifungal effects against the wood-degrading fungi Sphaeropsis sapinea and Trichoderma harzianum. Exposure to increasing concentrations of chitosan caused an increase in the amount of hydrogen peroxide accumulation in cultures of S. sapinea, which was accompanied by a decrease in superoxide formation. The same effect was not observed in T. harzianum. Potassium ion leakage was an early event for both test fungi, leakage being more pronounced for S. sapinea than T. harzianum for the first 5 min, particularly at higher concentrations of chitosan treatment. Fluorescence microscopy provided evidence that the effect of chitosan on fungal hyphae was mediated through alterations in the plasma membrane properties. Chitosan also severely affected fungal morphology. Increasing concentrations of chitosan induced excessive branching, vacuolation, and a reduction in hyphal diameter. Transmission electron microscopy, which showed more severe ultrastructural changes in S. sapinea hyphae from chitosan treatment as compared to T. harzianum, provided valuable complementary information. The data suggest that the plasma membrane may be the primary target of chitosan action, and that the two fungi differ in the extent to which they are affected.     

Effect of polymer differences on fungal penetration of hydrogel lenses

Summary Two chemically distinct types of hydrogel lenses, vifilcon A and bufilcon A, each with a water content of 55%, were challenged in a balanced salts solution withAspergillus fumigatus, Cladosporium cladosporioides, Curvularia lunata andFusarium solani. The lenses were cleaned, disinfected and stained after varying periods of incubation and examined with light microscopy and scanning electron microscopy. For three of the four fungi, the bufilcon A lens was more susceptible to fungal attack than the vifilcon A lens.Curv. lunata produced the greatest number of penetration pegs within 72 h for both lens types. Etching of lens surfaces was observed withC. cladosporioides. In general, the susceptibility of a hydrogel lens to penetration with a fungus appeared to vary with the species of fungus and the chemical composition of the lens.     

Antimicrobial activity of resin acid derivatives

The wide potential of resin acids as bioactive agents gave rise to a growing effort in the search for new applications of the natural forms and their derivatives. In some of these compounds, the antimicrobial activity is associated to the presence in the molecules of functional groups such as the hydroxyl, aldehyde, and ketone or to their cis or trans configurations. The resin acid family covers a spectrum of antimicrobial activities against several microorganisms, from bacteria to fungi, in which the mode of action was studied by electron microscopy. The morphological alterations are consistent with an unspecific mode of action causing inhibition of the fungal growth or damaging the fungal cells in parallel with a mechanism of resistance based on the retention of the compound by the lipid accumulation. The sterol composition of phytopathogenic fungi Botrytis cinerea and Lophodermium seditiosum treated with methyl cis-7-oxo-deisopropyldehydroabietate revealed the presence of ergosterol (M+ 396) and dihydroergosterol (M+ 398) in both cultures showing that this compound did not interfere with the ergosterol metabolic pathway of both fungi.     

Ultrastructural aspects of cytoplasmic ribosomes from Histoplasma capsulatum and Blastomyces dermatitidis as revealed by heavy metal staining

Cytoplasmic ribosomes were isolated and purified from sonicates of the mycelial and yeastlike growth forms of the pathogenic dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. Similar ribosomal fractions were prepared from Neurospora crassa and Saccharomyces cerevisiae. These latter organisms were selected as typical filamentous and yeastlike monophasic fungi, and their ribosomes were used as reference standards. High resolution electron microscopy permitted a comparison of both positively and negatively-stained ribosomes to those dehydrated without heavy metal salt. Such studies revealed statistically significant differences in physical dimensions. Cautious interpretations of substructural detail of the various ribosomal preparations suggested both interphasic and interspecies differences.     

Scanning electron microscopy of microspore embryogenesis inBrassica spp.

Scanning electron microscopy was employed to study and compare microspore embryogenesis in vitro with pollen development in planta inBrassica napus andB. oleracea. An exine with its specific pattern had already been formed, when microspores were released from tetrads. During subsequent pollen development, microspores increased in size and continued to strengthen the exine. Upon in vitro culture, all microspores, i.e., embryogenic and nonembryogenic, initially showed the same morphological features. After 24 h in culture, the microspores had increased in size. Thereafter, embryogenesis was indicated in some microspores by two different morphological changes. One featured an expansion in volume of the cell cluster around the germination aperture (type I), the other showed cell cluster volume expansion over the entire microspore surface (type II). Two-thirds of embryogenic microspores in bothB. napus andB. oleracea demonstrated type I development. When followed by fluorescence microscopy, in vitro culture of microspores revealed cultures with a high embryo frequency were those with a high frequency of symmetrical division.Abbreviations SEM Scanning electron microscopy - TEM Transmission electron microscopy