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.     

Antifungal potential of essential oil and various organic extracts of <Emphasis Type="Italic">Nandina domestica</Emphasis> Thunb. against skin infectious fungal pathogens

This study was undertaken to assess the in vitro antifungal potential of the essential oil and n-hexane, chloroform, ethyl acetate, and methanol extracts of Nandina domestica Thunb. against dermatophytes, the casual agents of superficial infections in animals and human beings. The oil (1,000 μg/disc) and extracts (1,500 μg/disc) revealed 31.1–68.6% and 19.2–55.1% antidermatophytic effect against Trichophyton rubrum KCTC 6345, T. rubrum KCTC 6375, T. rubrum KCTC 6352, Trichophyton mentagrophytes KCTC 6085, T. mentagrophytes KCTC 6077, T. mentagrophytes KCTC 6316, Microsporum canis KCTC 6591, M. canis KCTC 6348, and M. canis KCTC 6349, respectively, along with their respective minimum inhibitory concentration values ranging from 62.5 to 500 and 125 to 2,000 μg/ml. Also, the oil had strong detrimental effect on spore germination of all the tested dermatophytic fungi as well as concentration and time-dependent kinetic inhibition of T. rubrum KCTC 6375. The present results demonstrated that N. domestica mediated oil and extracts could be potential sources of natural fungicides to control certain important dermatophytic fungi.     

Biotic effect of fungal communities inhabiting grapevine phyllosphere on <Emphasis Type="Italic">Phoma negriana</Emphasis>

The effect of 15 fungi species most frequently inhabiting grapevine shoots on the growth of Phoma negriana was examined. Particular species-components of the fungal community were tested in vitro against the pathogen, using the biotic series method. The majority of fungi species inhibited P. negriana growth and the growth of only 3 species from the community, i.e. Alternaria alternata Keiss., Botrytis cinerea Pers. and Phomopsis viticola, was limited by the pathogen. Fungi Trichoderma were found the most effective against P. negriana. They completely degraded the hyphae and conidia of P. negriana after 20–22 days of growth in two-organism cultures. The summary biotic effects of fungal communities from grapevine were generally favourable to the P. negriana growth and only in one year of study they suppressed the pathogen development.     

Infectivity of two members of theEntomophthora muscae complex [Zygomycetes: Entomophthorales] forMusca domestica [Dipt.: Muscidae]

Dose-mortality studies were conducted with 2 members of theEntomophthora muscae (Cohn) Fresenius complex from southern California (CA) and Denmark (DA) infecting house flies,Musca domestica L., from southern California. Primary conidia of the DA form were significantly more infective (LC₅₀=34 conidia/mm²) than primary conidia of the CA form (LC₅₀=67 conidia/mm²) for 2–7 days old (‘young’) flies. Infectivity (single experimental trial) for 10–14 day old (‘old’) flies of primary conidia of the CA form (LC₅₀=34 conidia/mm²) was similar to that of the DA form for young flies. Secondary conidia of the CA form were markedly more infective (LC₅₀=0.36 conidia/mm²) than primary conidia of either form. Dose had no significant effect on incubation period for primary conidia of either form, but increased doses resulted in significantly shorter incubation periods for flies exposed to secondary conidia of the CA form.      

Cytolytic activity of Bacillus thuringiensis CryIC and CryIAc toxins to Spodoptera sp. midgut epithelial cells in vitro

Summary A sensitive lactate dehydrogenase (LDH) assay was modified to determine the cytolytic activity of Bacillus thuringiensis CryIC and CryIAc delta endotoxins to viable collagenase-dissociated midgut epithelial cells (MEC) from larvae of Spodoptera frugiperda and Spodoptera exigua. The MEC preparations from these Spodoptera sp. consisted predominantly of columnar cells (65–75%) and goblet cells (25–35%). Time course microscopy experiments indicated that only the columnar cells became swollen during CryIC toxin incubation. Also, comparative cytotoxicity studies were run with cell lines of nonmidgut origin established from S. frugiperda (SF21AE) and S. exigua (SEUCR1A). Optimum conditions for the cytotoxicity assay were similar for MEC and cell lines of both species, and were met in an assay in which 0.1-ml cell concentrations (8.5±0.5×10⁴ cells) were incubated with toxin dilutions (0.01–20 μg) for 1 h at 24° C at a final pH of 7.8. The Spodoptera sp. MEC were twofold more sensitive to CryIC (68% lysis) than CryIAc (32% lysis) at optimum toxin levels (2.5–5 μg). Also, the SEUCR1A cells were more sensitive (2.3-fold) to CryIC (70% lysis) than CryIAc (30% lysis) at optimum toxin levels of 5–10 μg. The SF21AE cells, however, were twofold less sensitive to CryIC (30% lysis) than SEUCR1A cells and response to CryIAc and CryIC was similar. Immunoblot analysis of either Spodoptera sp. MEC or brush border membrane vesicles (BBMV) identified seven CryIC binding proteins with molecular mass of 137, 120, 115, 68, 65, 63, and 45 kDa. Occasionally, a 148-kDa protein band was observed. The CryIAc toxin bound to two proteins on MEC and BBMV with molecular mass of 137 and 120 kDa.     

Use of clove oil to anaesthetize freshwater amphipods

The use of clove oil as a potential anaesthetic for freshwater amphipods was examined at 20 °C. Individuals of Gammarus minus, a common species in southern Illinois, USA, spanning the entire body size range (4.3–14.3 mm), were used to test four anaesthetic concentrations varying from 1.48 × 10⁻⁴ ml ml⁻¹ to 5.9 × 10⁻⁴ ml ml⁻¹. Small-bodied individuals (mean size = 5.4 mm ± 0.27SE) were used to test additional concentrations, up to 14.7 × 10⁻⁴ ml ml⁻¹, a 10-fold span, to identify potential lethal concentrations. At the lowest concentration, time to anaesthesia and recovery was constant at all body sizes. For the three next higher concentrations, time to anaesthesia decreased with increasing concentration while recovery time increased. Activity of amphipods was not affected by the ethanol carrier. In addition, activity did not differ between amphipods that had recovered from anaesthesia and unexposed amphipods. At clove oil concentrations of 8.84 × 10⁻⁴ ml ml⁻¹ and 14.7 × 10⁻⁴ ml ml⁻¹, mortality was 7 and 40%, respectively, indicating, that 5.9 × 10⁻⁴ ml ml⁻¹ was a safe working concentration. No mortality was observed with Gammarus acherondytes, a federally endangered cave amphipod on which the protocol with 80 μl of stock was used in the field. The method enabled us to obtain information on the endangered amphipod which normally would have required the sacrifice of individuals. Thus, research can continue on species for which population numbers are low and for which basic information is needed to formulate meaningful recovery plans.     

Inhibitory effect of essential oil from <Emphasis Type="Italic">Nepeta rtanjensis</Emphasis> on fungal spore germination

The ability of Nepeta rtanjensis essential oil to inhibit conidia germination of fungi was evaluated in vitro. Tested fungi included in research were Cladosporium cladosporioides, Trichoderma viride and two Alternaria species originally isolated from N. rtanjensis. The conidia of Cladosporium cladosporioides were most susceptible to the N. rtanjensis essential oil treatment, and the oil concentration of 0.1 μg ml⁻¹ caused the maximum of conidia germination inhibition. The highest concentration used in experiment that caused the maximum of conidia germination inhibition was 0.6 μg ml⁻¹ for Alternaria isolated from leaf surface of N. rtanjensis. The germ tube elongation of Alternaria isolates significantly decreased in response of different concentrations of oil used in experiment.     

Chemical composition and antimicrobial activity of the essential oil of Cacalia tangutica (Maxim.) Hand.-Mazz

Essential oil of the subterranean part of Cacalia tangutica (Maxim.) Hand.-Mazz was analyzed by gas chromatography (GC)-mass spectrum (MS) technique in two different capillary columns of different polarities. Thirty-one components were identified in the oil and the main compounds were α-zingiberene (13.49%), germacrene D (10.76%), α-pinene (8.54%), caryophyllene(Z-) (6.36%), linalool (6.16%), β-myrcene (4.89%), β-ocimene (Z-) (4.40%)and ocimenone(Z-) (3.58%). The antimicrobial activity of the oil was evaluated against 2 fungi and 12 bacteria including 6 clinically isolated strains using the agar disc diffusion and broth microdilution methods. The results show that the oil presented a broad antimicrobial spectrum and had better antimicrobial activity against yeast and gram-positive bacteria. The minimum inhibitory concentration values were 0.16–5.00 g/L and minimum bactericidal concentration values were 0.16–5.00 g/L. __________ Translated from Journal of Wuhan University (Science Edition), 2007, 53 (2): 198–203 [译自: 武汉大学学报(理学版)]     

Fruit-body production of two ectomycorrhizal fungi in the genusHebeloma in pure culture

Fruit-body production of two ectomycorrhizal fungi,Hebeloma radicosum andhebeloma sp. (nagaenosugitakedamashi in Japanese), in pure culture was examined. First, nutrients that promote mycelial growth of the fungi when added to the basal medium consisting of barley grains and sawdust were determined. Then the fungi were cultivated to produce fruit-bodies in larger-scale media containing additional nutrients selected for each fungus. Mature fruit-bodies bearing basidiospores were formed after incubation at 22°C for 35–42 d, followed by incubation at 17°C for 21–32 d.     

Effects of combining the fungi <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhiziumanisopliae</Emphasis> on the mortality of the tick <Emphasis Type="Italic">Amblyomma variegatum</Emphasis> (ixodidae) in relation to seasonal changes

The effect of formulation, fungal concentration, type and seasonal changes on the mortality of the tick Amblyomma variegatum was investigated. A previous study demonstrated high pathogenicity of strains of the fungi Beauveria bassiana and Metarhizium anisopliae against the ticks Rhipicephalus appendiculatus and Amblyomma variegatum (Kaaya et al. J Invertebr Pathol 1996; 67: 15–20). The present study was undertaken to explore possible additive or synergistic effects of the two fungi on A. variegatum. The effects of oil and water formulations at different concentrations of each fungus and combination of the two on the mortality of A. variegatum in the laboratory and in the field during the wet and dry seasons were determined and compared. The oil formulation performed better in all assays, with highest tick mortality of 92% occurring during the wet season at conidia concentration of 1 × 10¹⁰ conidia/ml of the mixed fungi compared to 49% for the water formulation at similar conidia concentration. However, at the same conidial concentration during the dry season, mortalities in the field were relatively low with the mixture of the fungi recording 24% and 17% tick mortality for the oil and water formulation respectively. The effect of infecting the ticks with a cocktail of the two fungi was inconclusive under more controlled conditions in the laboratory but field results under both wet and dry seasons indicated significant differences between the separate and mixed fungi infections. The results demonstrate a potential of cocktail formulations in the control of ticks and possibly of other arthropod pests.     

A viscometric study of the biodegradation of photographic gelatin by fungi isolated from cinematographic films

The biodegradation of photographic gelatin grade (Bloom 225) material was studied by viscometry in aqueous solution (at 37 °C, 6.67% w/w) using filamentous fungi isolated and identified from cinematographic film stored in different Spanish archives. From viscosity data, different variables such as molecular weight and chain scission were calculated. To ensure initial spore suspension concentration was standardized for all the biodegradation experiments, a correlation between transmittance at 530 nm of fungal spore suspensions and the corresponding cytometric determination of populations was established for all the fungal strains studied in this work. The bioassay experiments were carried out at 25 and 4 °C using an initial concentration of fungi of 4.5×10⁵ conidia/mL except in the case of the genus Alternaria, where the concentration was 10 times lower. The fungal strains were three species of Aspergillus, i.e., A .ustus, A. nidulans var. nidulans, A. versicolor, seven Penicillium chrysogenum strains, and Cladosporium cladosporioides, Alternaria alternata, Mucor racemosus, Phoma glomerata, and Trichoderma longibrachiatum. All were gelatinase positive. Through the viscosity decay profiles with bioassay-time and the corresponding calculated chain scission, the relative quantitative gelatinase efficiency of these fungi has been evaluated.     

四种植物精油对黑翅土白蚁触杀和驱避作用

为筛选出高效防治黑翅土白蚁的天然植物精油,减少有机合成农药的使用,该文研究了大蒜精油、肉桂油、丁香油和印楝素油四种植物精油对黑翅土白蚁的触杀效果和驱避作用。结果表明:大蒜精油、肉桂油和丁香油的浓度为5和10 mg·m L~(-1)时,处理2 h后,黑翅土白蚁的校正死亡率达100%,而相同浓度的印楝素油和对照处理的黑翅土白蚁校正死亡率低于5%。随着处理时间延长,浓度为1.25和2.5 mg·m L~(-1)的大蒜精油、肉桂油和丁香油处理6 h时,黑翅土白蚁的校正死亡率仍达100%,而此时对应的印楝素油和对照处理的黑翅土白蚁校正死亡率仅为10%,说明大蒜精油、肉桂油和丁香油对黑翅土白蚁具有较强的触杀效果。大蒜精油、丁香油和肉桂油在处理黑翅土白蚁2 h后LC_(50)值(半致死量)分别为1.572、1.05和1.03mg·m L~(-1),说明肉桂油对黑翅土白蚁的毒性相对最大,触杀效果最好。此外,10 mg·m L~(-1)的大蒜精油、肉桂油、丁香油和印楝素油的驱避试验表明,处理4、6、8和12 h后,大蒜精油、肉桂油和丁香油三精油处理区的黑翅土白蚁数均显著低于对照区的,驱避率总体93%,而对应的印楝素油的驱避率总体28.5%,表明大蒜精油、丁香油和肉桂油三种植物精油对黑翅土白蚁均有显著的驱避活性。综上可知,四种植物精油中大蒜精油、肉桂油和丁香油在防治黑翅土白蚁方面应用潜力很好,是开发绿色环保白蚁防治药剂的可选材料。     

Paddy straw as substrate for ethanol production

Pretreatment of paddy straw with 2% sodium hydroxide at 15 psi for 1 h resulted in 83% delignification. The hydrolysis of alkali treated paddy straw with a commercial preparation of cellulase for 2 h at 50°C resulted in release of 65% total reducing sugars. Maximum sugars were released at enzyme loading of 1.5% (v/v). The fermentation of hydrolysate supplemented with nutrients by S. cerevisiae resulted in the production of 20–30 g L⁻¹ ethanol after 48 h incubation which was further improved with addition of yeast nitrogen base and inoculated with 1% (w/v) yeast cells.     

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.     

New species ofAcremonium, Cylindrocarpon andVerticillium from soil in the Bonin (Ogasawara) Islands, Japan

Three new soil fungi from the Bonin (Ogasawara) Islands, Japan are described:Acremonium macroclavatum, characterized by large clavate guttulate conidia;Cylindrocarpon boninense, characterized by 3–7-septate clavate macroconidia, terminal or intercalary chlamydospores, rarely produced unicellular clavate microconidia; andVerticillium hahajimaense, characterized by conidial heads bearing cylindrical conidia, and catenulate chlamydospores.     

Effect of nitrogen source on lignocellulolytic enzyme production by white-rot basidiomycetes under solid-state cultivation

Summary The effect of additional nitrogen sources on lignocellulolytic enzyme production by four species of white-rot fungi (Funalia trogii IBB 146, Lentinus edodes IBB 363, Pleurotus dryinus IBB 903, and P. tuberregium IBB 624) in solid-state fermentation (SSF) of wheat straw and beech tree leaves was strain- and substrate-dependent. In general, the yields of hydrolytic enzymes and laccase increased by supplementation of medium with an additional nitrogen source. This stimulating effect of additional nitrogen on enzyme accumulation was due to higher biomass production. Only xylanase specific activity of P. dryinus IBB 903 and laccase specific activity of L. edodes IBB 363 increased significantly (by 66% and 73%, respectively) in SSF of wheat straw by addition of nitrogen source to the control medium. Additional nitrogen (20 mM) repressed manganese peroxidase (MnP) production by all fungi tested. The study of the nitrogen concentration effect revealed that 10 mM peptone concentration was optimal for cellulase and xylanase accumulation by P. dryinus IBB 903. While variation of the peptone concentration did not cause the change in MnP yield, elevated concentrations of this nutrient (20–40 mM) led to a 2–3-fold increase of P. dryinus IBB 903 laccase activity. About 10–20 mM concentration of NH₄NO₃ was optimal for cellulase and xylanase production by F. trogii IBB 146. However, neither the laccase nor the MnP yield was significantly changed by the additional nitrogen source.     

Composition and Nutritional Characteristics of Fungi Consumed by Callimico goeldii in Pando, Bolivia

Though ≥22 species of Primates consume fungi, most do so at low rates, comprising <5% of their feeding time. Goeldi's monkeys (Callimico goeldii), spend up to 29% of their feeding time year-round consuming fungal sporocarps, the fruiting bodies of fungi. We provide comprehensive data on the nutritional characteristics of 4 species of fungi consumed by Callimico goeldii (Ascopolyporus polyporoides, Ascopolyporus polychrous, Auricularia auricula, and Auricularia delicata). The composition of the fungi is similar to that of other fungi: predominantly fiber (66.2–83.0% dry matter) with small amounts of sugar (2.0–5.6% dry matter) and crude fat (0.9–1.6% dry matter). Though the crude protein content is substantial (5.5–13.4% dry matter), much of the nitrogen in the fungi is not likely to be available to Callimico goeldii because it is associated with indigestible food components or is in nonprotein form. The mineral content of the fungi are within the normal range for fungi generally and the calcium-to-phosphorus ratio is low (0.07–0.25). Fungi appear to be a low-quality food resource for Callimico goeldii and may contribute to their relatively large home ranges and low population density compared to other Callitrichinae. Research on the ability of Callimico goeldii to digest fungi is needed to understand fully the nutritional value of fungi to them. We discuss adaptations Callimico goeldii may have for improving their ability to obtain nutrients from fungi and potential ecological correlates of mycophagy.     

Measurement of particle diameter of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> microcapsule by spray drying and analysis on its microstructure

Lactobacillus acidophilus, as a probiotic, is widely used in many functional food products. Microencapsulation not only increases the survival rate of L. acidophilus during storage and extends the shelf-life of its products, but also optimal size microcapsule makes L. acidophilus have an excellent dispersability in final products. In this paper, L. acidophilus was microencapsulated using spray drying (inlet air temperature of 170°C; outlet air temperature of 85–90°C). The wall materials used in this study were β-cyclodextrin and acacia gum in the proportion of 9:1 (w/w), and microcapsules were prepared at four levels of wall materials (15, 20, 25 and 30% [w/v]) with a core material concentration of 6% (v/v). The microcapsule diameters were measured by Malvern’s Mastersizer-2000 particle size analyzer. The results showed that the particle diameters of microcapsule were mostly within 6.607 μm and 60.256 μm and varied with 2.884–120.226 μm (the standard smaller microcapsule designated as <350 μm). Through comparison of microcapsule size and uniformity with different concentration of wall materials, we concluded that the optimal concentration of wall material was 20% (w/v), which gave microcapsule with a relatively uniform size (averaging 22.153 μm), and the number of surviving encapsulated L. acidophilus was 1.50 × 10⁹ c.f.u./ml. After 8 weeks storage at 4°C, the live bacterial number was above 10⁷ c.f.u./ml, compared with unencapsulated L. acidophilus, 10⁴–10⁵ c.f.u./ml. Through the observation of scanning electron microscopy, we found that the shapes of microcapsule were round and oval, and L. acidophilus cells located in the centre of microcapsule.     

Molecular and morphological description of a new species of Ulocladium from Southern China

A new species of Ulocladium was discovered from diseased leaves of Lycopersicon esculentum and Duchesnea indica from Hunan Province of China. Morphologically, this species is very close to U. consortiale, U. cucurbitae, and U. subcucurbitae in producing narrow ellipsoid conidia at 1–3 days, but the conidial size range of this species at this stage could distinguish it from three well-known species. It also exhibits the multiplex conidium morphology at the different growth-stages (1–3 days and 4–7 days). The results of maximum parsimony (MP) and neighbor-joining (NJ) phylogenetic analyses of combined glyceraldehyde-3-phosphate dehydrogenase (gpd) gene and Alternaria alternata major allergen (Alt a 1) genes show that U. solani and U. subcucurbitae cluster in a unique and separate subclade with no clear affinities to a specific sistergroup, and demonstrate that the Ulocladium species group is monphyletic, but two clades of this section are recognized. Morphological features of this new species, the sequences of the Alt a 1 and gpd gene regions, and its comparison with related species in this genus are discussed.     

Acaricidal effects of <Emphasis Type="Italic">Corymbia citriodora</Emphasis> oil containing <Emphasis Type="Italic">para</Emphasis>-menthane-3,8-diol against nymphs of <Emphasis Type="Italic">Ixodes ricinus</Emphasis> (Acari: Ixodidae)

The toxicity of para-menthane-3,8-diol (PMD), the main arthropod-repellent compound in the oil of the lemon eucalyptus, Corymbia citriodora, was evaluated against nymphs of Ixodes ricinus using five methods (A–E) of a contact toxicity bioassay. Mortality rates were estimated by recording numbers of dead nymphs at 30 min intervals during the first 5 h after the start of exposure and at longer intervals thereafter. The mortality rate increased with increasing concentration of PMD and duration of exposure with a distinct effect after 3.5 h. From the results obtained by methods A, C and E, the LC₅₀ range was 0.035–0.037 mg PMD/cm² and the LC₉₅ range was 0.095–0.097 mg PMD/cm² at 4 h of exposure; the LT₅₀ range was 2.1–2.8 h and the LT₉₅ range was 3.9–4.2 h at 0.1 mg PMD/cm². To determine the duration of toxic activity of PMD, different concentrations (0.002, 0.01, 0.1 mg PMD/cm²) were tested and mortality was recorded at each concentration after 1 h; thereafter new ticks were tested. This test revealed that the lethal activity of PMD remained for 24 h but appeared absent after 48 h. The overall results show that PMD is toxic to nymphs of I. ricinus and may be useful for tick control.