香茅精油对桔小实蝇产卵驱避作用及其化学成份分析

研究了香茅Cymbopogon nardus精油对桔小实蝇Bactrocera dorsalis (Hendel)的产卵驱避作用及其化学成分。结果表明,香茅精油对桔小实蝇产卵具有一定的驱避作用,不同浓度香茅精油处理芒果上的产卵量均显著低于对照,且随着香茅精油浓度的提高其产卵量逐渐减少,当浓度为10000μg/mL时,产卵驱避率为70.06%。采用气相色谱—质谱联用(GC-MS)技术分析了香茅精油的化学成份,结果发现该精油挥发物中含量较高的是3-蒈烯,香茅醛,香叶醇,香茅醇,柠檬烯。     

Susceptibility of the bruchid Callosobruchus maculatus (Coleoptera: Bruchidae) and its parasitoid Dinarmus basalis (Hymenoptera: Pteromalidae) to three essential oils

The bruchid Callosobruchus maculatus (F.) causes major losses during the storage of seeds of Vigna unguiculata (Walp.) in West Africa. An endemic parasitoid, the pteromalid Dinarmus basalis (Rond.) reduces the increase in bruchid populations in stores and could be used for biological control. African farmers often introduce essential oils into granaries at harvest time. In Togo, essential oils were extracted from two Gramineae, Cymbopogon nardus (L.) and Cymbopogon schoenanthus (L.) and from a Lamiaceae, Ocimum basilicum (L.). The major components of these essential oils were citronellal in C. nardus, carene-2 and piperitone in C. schoenanthus and estragol in O. basilicum. Cymbopogon schoenanthus was the most toxic oil for C. maculatus adults. D. basalis adults were more susceptible to the three essential oils than the adults of their hosts C. maculatus. In the presence of cowpea seeds, the LC50s of the three essential oils were lower than in their absence, suggesting that the seeds may absorb a part of the volatiles. High doses of three essential oils slightly affected the survival of the fourth instar or the pupae of C. maculatus. This high survival was due to protection of larvae from volatiles by the surrounding seeds. The D. basalis were more affected by the oil volatiles than their hosts. Sub-lethal doses of essential oils reduced the duration of the adult life of both insect species and fecundity of the females. The differences in sensitivity of the host and its parasitoid could influence their population dynamics. The introduction of the essential oils into storage systems potentially could reduce density of parasitoid populations and increase seed losses.     

Effects of Citrus sinensis (L.) Osbeck epicarp essential oil on growth and morphogenesis of Aspergillus niger (L.) Van Tieghem

Essential oils from different plant parts are known for their antimicrobial activity but the antifungal effects of essential oil from Citrus sinensis (L.) Osbeck epicarp on growth and morphogenesis of Aspergillus niger has not been observed so far. The mycelial growth was inhibited at 2.5 and 3.0 μg/ml of oil in Potato Dextrose Broth and Agar medium, respectively. These concentrations were fungicidal under the test conditions. The fungitoxicity of oil did not change even at exposure to 100 °C and autoclaving. The main changes observed under light and scanning electron microscopy after oil treatment were loss of cytoplasm in fungal hyphae, and budding of hyphal tip. The hyphal wall and its diameter became markedly thinner, distorted and resulted in cell wall disruption. The flattened and empty hyphal tips bifurcated into bud like structures. GC-MS studies of the oil revealed the presence of 10 chemical constituents. Limonene has been found to be major component (84.2%).     

香茅精油对番茄早疫病菌的抑菌作用及抑菌机制

为探明香茅精油的抑菌作用及其在植物病害生物防治中的应用价值,采用平板抑菌法和熏蒸法测定了香茅精油对番茄早疫病菌的抑菌活性,及其对菌丝体内电解质渗漏、可溶性蛋白质含量、丙二醛(MDA)含量、营养物质的吸收和超氧化物歧化酶(SOD)活性的影响.结果表明: 香茅精油对番茄早疫病菌有较强的抑菌作用,且该作用具有时间-剂量依赖性,但无杀菌作用.采用熏蒸法处理的抑制效果较平板抑菌法更好,处理48 h后,半抑制浓度(IC₅₀)分别为13.40 μL·L^-1和103.23 mg·L^-1;处理144 h后,IC₅₀分别为33.81 μL·L^-1和145.16 mg·L^-1.125 mg·L^-1香茅精油处理12 h后,菌丝体的电导率和MDA含量分别为对照的2.7和2.2倍,SOD活性和可溶性蛋白质含量分别提高88.5%和21.9%,还原糖的吸收减少11.3%.香茅精油可通过破坏病原菌细胞膜完整性和抑制菌体对营养物质的吸收来抑制病原菌菌丝的生长.香茅精油在植物病害生物防治中有一定的开发潜力.     

In vitro antifungal, anti-elastase and anti-keratinase activity of essential oils of Cinnamomum-, Syzygium- and Cymbopogon-species against Aspergillus fumigatus and Trichophyton rubrum

This study was aimed to evaluate effects of certain essential oils namely Cinnamomum verum, Syzygium aromaticum, Cymbopogon citratus, Cymbopogon martini and their major components cinnamaldehyde, eugenol, citral and geraniol respectively, on growth, hyphal ultrastructure and virulence factors of Aspergillus fumigatus and Trichophyton rubrum. The antifungal activity of essential oils and their major constituents was in the order of cinnamaldehyde>eugenol>geraniol=C. verum>citral>S. aromaticum>C. citratus>C. martini, both in liquid and solid media against T. rubrum and A. fumigatus. Based on promising antifungal activity of eugenol and cinnamaldehyde, these oils were further tested for their inhibitory activity against ungerminated and germinated conidia in test fungi. Cinnamaldehyde was found to be more active than eugenol. To assess the possible mode of action of cinnamaldehyde, electron microscopic studies were conducted. The observations revealed multiple sites of action of cinnamaldehyde mainly on cell membranes and endomembranous structures of the fungal cell. Further, test oils were also tested for their anti-virulence activity. More than 70% reduction in elastase activity was recorded in A. fumigatus by the oils of C. verum, C. martini, eugenol, cinnamaldehyde and geraniol. Similar reduction in keratinase activity in A. niger was recorded for the oils of C. martini and geraniol. Maximum reduction (96.56%) in elastase activity was produced by cinnamaldehyde whereas; geraniol caused maximum inhibition (97.31%) of keratinase activity. Our findings highlight anti-elastase and anti-keratinase activity of above mentioned essential oils as a novel property to be exploited in controlling invasive and superficial mycoses.     

Biosorption of Baftkar textile effluent

Decolourization of wastewater from a textile plant by a marine Aspergillus niger was studied. The fungus was previously isolated from Gorgan Bay in the Caspian Sea. The kinetics of decolourization was studied by varying energy sources. The best decolourization was achieved when sucrose was used as source of carbon and energy. NH4+ ion was demonstrated to be the best nitrogen source. Color reduction was found to increase from 80-97% as inoculum concentration increased from 0.04-1.0 g/L. A minimum inoculum of 0.2 g/L is necessary to achieve decolourization. The optimal temperature for the growth of A. niger on Baftkar wastewater is found to be 30 degrees C. 90-96% colour reduction is achieved in 19-20 hr of contact of mycelium cell with the wastewater. Colour reduction in a continuous column reactor of 70% was obtained using treated mycelium (NaOH, 90 degrees C) after 1 hr.     

Fungal Pathogens Associated with Banana Fruit in Sri Lanka,and their Treatment with Essential Oils

The crown rot pathogens isolated from banana samples collected from 12 localities in Sri Lanka were Lasiodiplodia theobromae, Fusarium proliferatum and Colletotrichum musae. Fungal pathogens isolated were able to cause crown rot disease alone or in combination. Disease severity was higher when combinations of virulent pathogens were used. Cymbopogon nardus and Ocimum basilicum oils displayed fungicidal activity against C. musae and F. proliferatum between 0.2-0.6% (v/v) in a Poisoned food bioassay. Slightly lower concentrations of the test oils were needed for similar activity during liquid bioassays. The combination of Cymbopogon nardus and O. basilicum oils demonstrated synergistic action during both in-vivo bioassays.     

Alteration of cell-wall composition ofFusarium oxysporum by copper stress

A strain ofFusarium oxysporum tolerated copper in the growth medium at concentrations up to 600 mg/L. The optimum growth was obtained at 200 mg Cu/L. The mycelium acquired a blue color in the presence of copper. The copper content of isolated cell walls obtained from mycelium grown in the presence of 600 mg Cu/L was 1.5 times higher than that of cell walls obtained from mycelium grown at 200 mg Cu/L and it contained 2.2 and 3.3% copper at 200 and 600 mg Cu/L, respectively. The amount of protein and total sugars increased in both the mycelium and its isolated cell walls in the presence of copper in the growth medium, chitin was also increased in the cell wall, reaching its maximum amount at 200 mg Cu/L— about 2.4 times higher than without copper. Most of amino acid concentrations in the cell wall were increased in the presence of 200 mg Cu/L and decreased above this concentration. Isoleucine, leucine, tyrosine, phenylalanine, and arginine showed the highest increase at this concentration. The altered cell walls obtained from mycelium grown at 200 and 400 mg Cu/L could rebind individual metals more than the control cell walls could. Rebinding of individual metals was in the order Zn>Fe>Ni>Cu>Co. Rebinding of copper by isolated cell walls depended on pH and temperature.     

Ultrastructural localization of heavy metals in the extraradical mycelium and spores of the arbuscular mycorrhizal fungus Glomus intraradices

Arbuscular mycorrhizal fungi, obligate symbionts of most plant species, are able to accumulate heavy metals, thereby, protecting plants from metal toxicity. In this study, the ultrastructural localization of Zn, Cu, and Cd in the extraradical mycelium and spores of the arbuscular mycorrhizal fungus Glomus intraradices grown in monoxenic cultures was investigated. Zinc, Cu, or Cd was applied to the extraradical mycelium to final concentrations of 7.5, 5.0, or 0.45 mmol/L, respectively. Samples were collected at time 0, 8 h, and 7 days after metal application and were prepared for rapid freezing and freeze substitution. Metal content in different subcellular locations (wall, cytoplasm, and vacuoles), both in hyphae and spores, was determined by energy-dispersive X-ray spectroscopy. In all treatments and fungal structures analysed, heavy metals accumulated mainly in the fungal cell wall and in the vacuoles, while minor changes in metal concentrations were detected in the cytoplasm. Incorporation of Zn into the fungus occurred during the first 8 h after metal addition with no subsequent accumulation. On the other hand, Cu steadily accumulated in the spore vacuoles over time, whereas Cd steadily accumulated in the hyphal vacuoles. These results suggest that binding of metals to the cell walls and compartmentalization in vacuoles may be essential mechanisms for metal detoxification.     

Live-cell imaging of vegetative hyphal fusion in Neurospora crassa

The process of hyphal fusion (anastomosis) in growing colonies of Neurospora crassa, stained with the membrane-selective dyes FM1-43 and FM4-64, was visualized by confocal microscopy. Time-lapse, live-cell imaging illustrated the dynamics of hyphal growth and anastomosis during its pre-contact, contact and post-contact, and post-fusion stages. Fusion-competent hyphae were morphologically distinct and exhibited remote sensing, resulting in branch initiation and/or re-direction of growth to facilitate contact between participating hyphae. A stained Spitzenk?rper was often observed where fusion-competent hyphae met. It is suggested that this structure contains secretory vesicles responsible for the delivery of cell adhesion molecules at the point of contact, cell wall synthesizing enzymes for the swelling growth of fused hyphal tips, and digestive enzymes required for fusion pore formation. Dramatic changes in cytoplasmic flow frequently occurred between the participating hyphae following fusion. After anastomosis has taken place, septa commonly formed close to the fusion site. The live-cell imaging reported here has clearly shown the complexity of the hyphal homing and fusion process. The control and consequences of repeated anastomoses within a mycelium must be as complex as the process itself.     

Response of Java citronella (Cymbopogon winterianus Jowitt) to toxic heavy metal cadmium.

Cadmium at 200 mg kg-1 soil and above concentrations was fatal as growth was inhibited ultimately leading to death of Java citronella (Cymbopogon winterianus Jowitt.). The surviving plants at 50 and 100 mg kg-1 treatments also exhibited pronounced retardation of growth and biomass yield. There was considerable reduction in the level of essential oil in herbage and oil quality deteriorated. Cadmium accumulation profile showed that highest accumulation was in root, followed by stem, leaf sheath and leaf. Very high accumulation in root for higher doses appeared to be the reason for fatality.     

铅、锰对黄伞菌丝形态及细胞活性的影响

【目的】确定铅(Pb2+)、锰(Mn2+)对黄伞菌丝形态、结构及其菌丝活力的影响,并比较黄伞对Pb2+、Mn2+的适应性和耐受性。【方法】采用平板培养和液体培养方法,结合菌落形态和菌丝的电镜观察,测定菌落直径、菌丝鲜重,并以原子吸收分光光度计测定Pb2+、Mn2+含量。同时测定液体培养条件下菌丝的鲜重和胞外多糖产量以验证菌丝活力。【结果】不同Pb2+、Mn2+浓度下黄伞菌丝体形态变化明显,Pb2+、Mn2+的浓度≥500 mg/L可显著抑制黄伞菌丝的生长;高浓度Pb2+、Mn2+下(Pb2+≥700 mg/L、Mn2+≥2 000 mg/L)黄伞菌丝体锁状联合大量减少,且大小不一、分布不均,菌丝褶皱变形。黄伞在菌丝平板生长过程中,当Pb2+、Mn2+分别为100 mg/L时,黄伞菌丝生长速度最快。黄伞在液体培养过程中,当Mn2+浓度为300 mg/L、Pb2+浓度为50 mg/L时,菌丝鲜重以及产生的胞外多糖含量最大。【结论】Pb2+、Mn2+对黄伞的菌丝生长、菌丝活力及结构形态有较大的影响;黄伞对锰离子的适应性和耐性明显高于对铅离子的适应性和耐性。     

Interactions of Gliocladium virens with Rhizoctonia solani and Pythium ultimum in Non-sterile Potting Medium

Interactions of Gliocladium virens with Pythium ultimum and Rhizoctonia solani under simulated in vivo conditions were observed microscopically. Different types of propagules of the three fungi were paired on nitrocellulose membranes and incubated at 25°C in non-sterile potting medium in Petri dishes for 1-5 days. Alginate-wheat bran prill were used as carriers for G. virens. Prill inoculated with G. virens and pre-incubated in potting medium for 3-5 days before placement on membranes did not inhibit the germination of Pythium sporangia, but subsequent Pythium growth was markedly stunted and distorted, with some hyphal collapse and cytoplasmic leakage. G. virens had no visible effect on older Pythium mycelium. Two to 5 days' growth of G. virens caused cytoplasmic leakage of Rhizoctonia mycelium, prevented secondary branching of hyphae and occasionally coiled around Rhizoctonia hyphae. Prill that were newly colonized by G. virens, but not prill pre-incubated for 3 or 5 days, stimulated the growth of Pythium mycelium and sporangia, Rhizoctonia mycelium and unprimed monilioid cells, probably by supplying nutrients. The timing of the interactions and their specificity for the different pathogen propagules were consistent with the production of gliotoxin by G. virens. This view was supported by in vitro experiments, in which pathogen propagules were incubated in a range of concentrations of gliotoxin in potato dextrose broth. Pythium sporangia and mycelium were inhibited by 1 or 2 μmg ml^-1, but Rhizoctonia monilioid cells and mycelium required 3-5 μmg ml^-1 for inhibition. At the lowest effective concentrations the inhibition was sometimes reversible, but propagules were killed at high concentrations of gliotoxin.     

In vitro studies of Coelomomyces punctatus from Anopheles quadrimaculatus and Cyclops vernalis

Attempts to grow mycelium of Coelomomyces punctatus from Anopheles quadrimaculatus larvae were made using more than 50 combinations of known vertebrate and invertebrate tissue culture media and microbiological media. Growth and/or differentiation of mycelium into sporangia were observed in several media. Significant growth of hyphal fragments and differentiation into young resting sporangia occurred in conditioned Mitsuhashi-Maramorosch insect tissue culture medium. This medium was conditioned by growth for 3 weeks in it of Varma's Anopheles stephensi tissue culture cells and was supplemented with 20% heat-inactivated fetal bovine serum and a synthetic tripeptide, glycyl-histidyl-lysine. Limited growth and elongation of lateral hyphal branches and subsequent development into resting sporangia with typical outer wall markings and pigmentation of mature forms were observed in a modified brain-heart infusion medium. Some media stimulated hyphae to develop into smooth-walled, spherical bodies of size and appearance typical of young sporangium initials but with no further maturity. In most media, no growth or development of mycelium occurred, but the fungus remained alive for 2–4 weeks. Mycelium of C. punctatus dissected from Cyclops vernalis did not grow and develop in any of the media utilized. However, in one case the mycelium differentiated into gametes shortly after dissection into modified brain-heart infusion medium.     

Localization of beta-(1,3)-glucanase in the mycelium of Sclerotium rolfsii.

The role of the lytic enzyme beta-(1,3)-glucanase in cell wall synthesis and its distribution in the mycelium of the fungus Sclerotium rolfsii were studied. Enzyme activity was determined after enzyme extraction with Triton X-100 from a cell wall preparation. Specific zones of immunofluorescence appeared in the hyphal tips, clamp connections, new septa, and lateral branching when a specific antiserum was used with the indirect method of the fluorescent antibody staining. Enzymatic activity in the cell wall preparation was inactivated by diethylpyrocarbonate. However, 69% of the total enzymatic activity was present in a latent form which was not affected by the ester. This result suggests that most of the beta-(1,3)-glucanase was present along the hyphal cell walls in a "masked" form. An active enzyme appeared only in those regions which showed immunofluorescence. The activity of glucan synthetase, an enzyme essential for wall formation, was higher in the branching funus grown on L-threonine-supplemented synthetic medium than in the synthetic medium-grown fungus.     

Zinc ions alter morphology and chitin deposition in an ericoid fungus

A sterile mycelium PS IV, an ascomycete capable of establishing ericoid mycorrhizas, was used to investigate how zinc ions affect the cellular mechanisms of fungal growth. A significant reduction of the fungal biomass was observed in the presence of millimolar zinc concentrations; this mirrored conspicuous changes in hyphal morphology which led to apical swellings and increased branching in the subapical parts. Specific probes for fluorescence and electron microscopy localised chitin, the main cell wall polysaccharide, on the inner part of the fungal wall and on septa in control specimens. In Zn-treated mycelium, hyphal walls were thicker and a more intense chitin labelling was detected on the transverse walls. A quantitative assay showed a significant increase in the amount of chitin in metal-treated hyphae.     

Ultrastructure of Mycoparasitism of Trichoderma, Gliocladium and Laetisaria Species on Botryodiplodia theobromae

Mycoparasitic activities of various isolates of Trichoderma viride, T. harzianum, T. hamatum, T. longibrachiatum, T. koningii, T. pseudokoningii, Gliocladium virens and Laetisaria arvalis were studied against a serious plant pathogen, Botryodiplodia theobromae by scanning electron microscopy (SEM). Macroscopic observations of fungal growth in dual-cultures revealed that most of the isolates made hyphal contact with the pathogen within 2 days after inoculation, leading to the inhibition in pathogen growth. However, T. viride Tv-4, T. hamatum and T. pseudokoningii inhibited pathogen growth before hyphal contact and exhibited an inhibition zone between the colonies of both fungi. SEM investigations demonstrated that in case of hyphal interaction, the firm binding of antagonists (T. viride Tv-1 & Tv-3, T. harzianum Th-1 & Th-2, T. longibrachiatum and L. arvalis) to B. theobromae hyphae established either by coiling around its hyphae, or by penetrating its hyphal cells by forming hooks, haustoria and appressoria-like structures which invariably led to cell disruption. Although T. koningii and G. virens Gv-2 & Gv-3 did not interact physically by way of coiling and penetration, they produced wall lytic enzymes or antifungal substances after coming in contact with B. theobromae which caused wrinkling, bursting and collapsing of pathogen mycelium. It is, therefore, suggested that the outcome of the interaction of antagonist and pathogen was most likely determined by initial hyphal contact that triggered a series of events in pathogen degradation.     

The effect of essential oil of Ammoides pusilla (brot.) breistr on the growth and the production of solanapyrone a by Ascochyta rabiei

Biological control such as the use of plant extracts has emerged as promising option to the phenomena of fungi resistance to chemical. Several constituent of essential oil have been studied for their biological activity including antibacterial and antifungal activity. In this study the effect of Ammoides pusilla essential oil with different concentrations was test against the growth of Ascochyta rabiei and the production of solanapyrone A by the fungus. After 14 days the mycelium was collected and the dry weight measured. A. rabiei did not grow at a final concentration of 6 and 3 mg/ml, at 1.5 mg/ml and 0.625ml there was little growth of the fungus with a dry weight of 55 mg and 99 mg respectively compared to the control with 519 mg dry weight, but there was no solanapyrone A produced. However a new compound appeared at the HPLC at 10 min. 30 sec. compared with the solanapyrone A which elutes at nearly 14 minutes.     

Antagonistic effect of fluorescent pseudomonads against Macrophomina phaseolina that causes charcoal rot of groundnut

Maximum colony growth inhibition was observed due to Pseudomonas PS2 (74%) as compared to PS1 (71%) on trypticase soy agar (TSM) plates after 5 days of incubation. Light and scanning electron microscopic examination showed hyphal coiling, vacuolation, coagulation and granulation of cytoplasm resulting in lysis of hyphae of M. phaseolina by pseudomonads. Cell free culture filtrates of strains PS1 and PS2 restricted the growth of mycelium of M. phaseolina. PS1 and PS2 caused maximum colony growth inhibition by 57 and 61% respectively at 20% concentration of culture filtrate after 4 days of incubation. Volatile substances produced by PS1 and PS2 also inhibited the colony growth of M. phaseolina by 25 and 32%, respectively. Inhibitory effect of volatile substances, however, decreased with advancing in incubation period. Colony growth of M. phaseolina was significantly decreased by PS1 and PS2 as compared to control both in iron- sufficient and iron-deficient conditions. PS2 showed higher antagonistic activity than PS1, as evidenced by pronounced colony growth inhibition.     

Morphometric evaluation of the specific growth rate of Aspergillus niger grown in agar plates at high glucose levels

Development of surface grown cultures of Aspergillus niger no. 10 was studied at two experimental levels: (a) following the time course of the biomass density (X [=] mg cm(-2)) and fitting the data by the logistic expression, which yielded a macroscopic specific growth rate expressed as mu(obs) = (dX/Xdt)[1-(X/X(max))](-1); and (b) measuring morphometric parameters like the specific elongation rate (k) of the germ tubes and their diameters (D(h)), the colony rate of radial extension (u(r)), and the mean length of distal hyphae (L(av)) to estimate the specific growth rate with the following proposed expression: mu(calc) = u(r)ln2[L(av)ln(L(av)/D(h))](-1). Increases in the initial glucose concentration (10, 40, 70, 120, 200, and 300 g L(-1)) caused reductions in the specific growth rates, the elongation kinetics of the germ tubes, and the hyphal diameter, nevertheless, u(r) and X(max) presented parabolic behavior, showing their maxima in the interval of 90 to 120 g L(-1) of glucose. The overall macroscopic effect of the tested concentrations of glucose on surface grown cultures of A. niger was to produce densely packed and slowly extending colonies, where changes in hyphal lengths and diameters were significant. There was good agreement between mu(obs) and mu(calc) values. Hence, this work validates a kinetic model based on morphometric data to estimate the specific growth rate of molds, obtained from dry weight data, using mold cultures grown in the same solid medium i.e., agar plates. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 287-294, 1997.