The effect of solanapyrone a produced by Ascochyta rabiei on seed germination and the elongation of radicles and hypocotyls of chickpea (Cicer areitinum L.)

Three Algerian isolates of A. rabiei (72, Mat 1.2 and 9216) were grown on Czapek Dox medium supplemented with cations and incubated for 14 days. After incubation, the mycelium of the fungus was removed by filtration through four layers of muslin cloth and spores were removed from the filtrate by centrifugation at 10,000 g for 20 min. Solanapyrone A was partially purified by liquid phase extraction into ethyl acetate and, after removal of the ethyl acetate, the toxin samples were dissolved in methanol and quantified by analytical High Performance Liquid Chromatography (HPLC). Solanapyrone A was identified by superimposition of its UV spectrum, obtained from the diode array detector of the HPLC, on the spectrum of an authentic sample. The action of solanapyrone A solution on seed germination and elongation of radicles and hypocotyls was tested using a concentration of 18.2 microg/ml and a two-fold dilution series of this solution in distilled water. The three Isolates, 72, Mat1.2 and 9216 produced solanapyrone A at concentrations of 37.2, 14.2 and 11.09 microg/ml, respectively. When probit % inhibition of seed germination was plotted against log2 of solanapyrone A concentration, there was a linear relationship and the EC50 concentration was determined as 7.2 microg/ml. Similarly, when radicle and hypocotyl elongation was plotted against log2 of solanapyrone A concentration, both gave linear relationships and the EC50 concentrations were determined as 5.37 and 6.02 microg/ml, respectively. It was concluded that solanapyrone A has a considerable inhibition of chickpea. However radicles and hypocotyls were susceptible than seed germination.     

Production of Ascochyta rabiei lacking solanapyrone A toxin production

Ascochyta rabiei, agent of Ascochyta blight of chickpea produces three toxins, Solanapyrones A, B and C of which solanapyrone A is the most toxic. All isolates of the fungus so far examined produce at least one of the Solanapyrone toxins, usually Solanapyrone A. The universality of solanapyrone production argues strongly for the importance of the toxins in virulence or pathogenicity. However, further evidence for this awaits the development of mutants lacking toxin production. Generation and isolation of fungal mutants defective in pathogenicity has been very useful for understanding the genetic and enzymatic processes responsible for infectivity in a number of pathosystems. Numerous tools have been used to transform plants and micro-organisms but the most widely micro-organism employed is Agrobacterium tumefaciens. In the present experiments, two strains of A. tumefaciens, AGL1 and LBA1126, harbouring two different plasmids, both encoding a gene for hygromycin resistance in the T-DNA region were used to transform isolate Tk21 of A. rabiei. The transformation of Ascochyta rabiei, gave rise to 498 colonies which grew on media supplemented with the selective agent; hygromycin B. The 30 sporulated transformants produced solanapyrone A on the specific medium at different rates. Solanapyrone A production, as demonstrated by the absorption of light at 327 nm, varied from 2.11 microg/ml to 4.32 microg/ml, representing a reduction of 74.11% to 46.99% in comparison with the wild type (8.15 microg/ml).     

Chemical composition, antioxidant and antimicrobial activities of essential oil of Thymus algeriensis wild-growing in Libya

The composition of essential oil isolated from Thymus algeriensis growing wild in Libya was analyzed by GC and GC-MS. The essential oil was characterized with thymol (38.50%) as the major component. The oil was screened for antioxidant activity using DPPH assay, and compared to thymol and carvacrol. Antioxidant activity was high, with the IC₅₀ of 0.299 mg/ml, compared to 0.403 and 0.105 mg/ml for thymol and carvacrol, and 0.0717 mg/ml for BHA. In addition, antimicrobial activity was tested against eight bacteria and eight fungi. T. algeriensis oil showed inhibitory activity against tested bacteria at 0.001–0.05 mg/ml, while bactericidal activity (MBC) was achieved at 0.0025–0.05 mg/ml. For antifungal activity MICs ranged 0.0005–0.025 mg/ml and MFC 0.001–0.05 mg/ml. High antimicrobial activity against the fungi in particular suggests that the essential oil of Thymus algeriensis could have a useful practical application.     

Studies in the mineral nutrition of Japanese mint

Summary Potassium deficiency resulted in a increased accumulation of total phosphorus, inorganic phosphorus and sugar phosphate which suggested some interference in phosphorus metabolism predominantly beyond the sugar phosphate synthesis stage. Roots appeared to be a reservoir for accumulation of various phosphorus fractions of Japanese mint (Mentha arvensis L. var.piperascens).A higher respiration rate under potassium deficiency might be one of the factors to retard the inclusion of inorganic phosphorus into structural components, resulting in poor formation of organophosphorus compounds needed for plant growth and development. In spite of an increased essential oil content (ml/100 g dry weight) in a phosphorus deficient situation, total essential oil production (ml/plant) was low. Foliage growth, essential oil content and respiration was higher between 60 to 80 days and seemed to be associated with a high intensity of enzymatic and metabolic activity, as observed in various phosphorus fractions rising to a very high value after 80 days of crop growth.     

Filamentous fungal growth assay: correlation between [U-14C] glucose uptake and dry weight determinations

The filamentous fungus Trichophyton mentagrophytes was grown in [U-14C] glucose supplemented nutrient broth. Growth was monitored in 200 microliter microcultures and 5 ml macrocultures by measuring the incorporation of 14C into trichloroacetic acid-insoluble macromolecules and in macrocultures by dry weight determination. Adjustment of the specific activity of the [U-14C] glucose medium supplement from 1080 muCi/mmole to 108 muCi/mmole decreased the rate of isotope uptake and prolonged availability so that growth beyond 36 h could be monitored radiometrically. The dry mycelial weight of the fungus was directly proportional (r = 0.995) to the amount of isotope incorporated. Isotope uptake in microcultures and macrocultures was kinetically identical. These data clearly indicate that the uptake of 14C by T. mentagrophytes can be used to monitor fungal growth accurately in both macrocultures and microcultures.     

高效降解石油外生菌根真菌的室内筛选

对7个外生菌根真菌菌株在不同石油浓度培养基中的生长及其对石油的降解作用进行了研究。结果表明:(1)不同菌株在同一石油浓度培养基中生长速度不同,同一菌株在不同石油浓度培养基中的生长速度亦不同。菌株010和菌株025在不同石油浓度培养基中生长速度均较其他菌株快。菌株010的菌丝干重随着石油浓度的增加而增加,当石油质量浓度为500 mg/L时,菌丝干重达到最大值,高于对照5.27%,石油对其生长产生了促进作用;当石油质量浓度为700 mg/L时,菌丝干重低于对照,随着石油质量浓度的升高,菌株生长呈下降趋势。石油质量浓度为100 mg/L时,菌株025生长最慢,菌丝干重低于对照9.1%。随着石油浓度的增加,菌株生长加快,当石油质量浓度为500 mg/L时,菌丝干重高于对照;当石油质量浓度为700 mg/L时,菌株025菌丝干重最高,高于对照25.65%。随着石油浓度的再度升高,菌株生长呈下降趋势。(2)不同菌株在同一石油浓度下对石油的降解能力不同,同一菌株在不同石油浓度下对石油的降解能力亦不同。菌株025对石油的降解能力最强,对石油的降解能力随着石油浓度的升高而提高。当培养基中石油质量浓度为900 mg/L时,菌株025对石油的降解率最高,达到73.65%。(3)菌株0100、09、035、LH004的菌丝生物量与对石油的降解能力呈正相关。     

Screening for inhibitory activities of essential oils on the growth of Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., the causal agent of leaf spot disease of Murraya koenigii L.

Seven essential oils namely clove, cedar wood, lemongrass, peppermint, eucalyptus, citronella and neem oils were tested for their inhibitory effect on spore germination, growth of germ tube and mycelial growth of Colletotrichum gloeosporioides isolated from diseased Murraya koenigii. All essential oils inhibited the germination and growth of germ tube at different concentrations. However, significant reduction in colony growth was observed with citrus, lemongrass and peppermint oils at 1000, 1500 and 2000 ppm concentrations, respectively. Citrus oil at 1360 ppm inhibited the maximum growth of the fungus followed by lemongrass oil at 1720 ppm and peppermint at 2260 ppm, respectively. The effect of essential oils on mycelial dry weight also showed antifungal activity on the growth of Colletotrichum gloeosporioides. The study revealed the possible utilisation of these essential oils for foliar spray for the management of leaf spot disease of Murraya koenigii.     

Inhibition of coagulase activity and growth ofStaphylococcus aureus by garlic extracts

Garlic extract (1 mg dry weight/ml) produced an inhibition of the coagulase reaction and increased the time of coagulation by a factor of 1.5, whereas 4 mg dry weight/ml increased the coagulation time factor by 2.75. At this latter concentration of garlic, coagulation was only partially complete at 4 h. Garlic extract (1.4 mg dry weight/ml) reduced growth in nutrient broth whereas 5.6 mg dry weight/ml was completely inhibitory. These effects were not observed until 8 h after exposure of the organisms to the garlic extracts. There were 5.9% more survivors among garlic treated mice compared to non-treated animals, but this difference is not significant.     

Reduction in solanapyrone phytotoxin production by Ascochyta rabiei transformed with Agrobacterium tumefaciens

Agrobacterium tumefaciens was used to transform Ascochyta rabiei, the causal agent of chickpea blight. Employing a T-DNA containing a hygromycin resistance gene (hph), 908 transformants were obtained from germinated pycnidiospores on a selective medium containing hygromycin. Transformants were confirmed using PCR and Southern analyses and of four of these that were tested, two had integrated multicopies of the hph gene, one had two copies and one had a single insertion. Transformants were tested for the production of solanapyrone A toxin using a microtitre plate assay. Loss of toxin production by transformants was confirmed by reversed phase high-performance liquid chromatography. Sixteen transformants out of 668 tested produced significantly less solanapyrone A than the wild-type strain.     

Chilli rhizosphere fungus Aspergillus spp. PPA1 promotes vegetative growth of cucumber (Cucumis sativus) plants upon root colonisation

A rhizosphere fungus was isolated from roots of chilli plants and identified as Aspergillus spp. PPA1. The fungus was tested for its ability to promote the growth of cucumber plants in a pot experiment. Cucumber seeds were sown in sterilised field soil amended with wheat grain inoculum (WGI) of PPA 1 at the rate of 0.5, 1 and 1.5% w/w, and plants were grown for 21 days in a net house. The treatment with PPA1 significantly increased shoot length, shoot fresh weight, shoot dry weight, root length, root fresh weight, root dry weight, plant length, leaf area and leaf chlorophyll content of cucumber plants compared to non-treated control. The growth promotion rate increased with the increasing concentration of inoculum of PPA1 applied to the soil. The fungus was re-isolated from the roots of cucumber plants at higher frequencies. These results suggest that Aspergillus spp. PPA1 is a root colonising plant-growth promoting fungus for cucumber.     

Improvement of the conversion of polystyrene to polyhydroxyalkanoate through the manipulation of the microbial aspect of the process: a nitrogen feeding strategy for bacterial cells in a stirred tank reactor

Pseudomonas putida CA-3 has been shown to accumulate the biodegradable plastic polyhydroxyalkanoate (PHA) when fed styrene or polystyrene pyrolysis oil as the sole carbon and energy source under nitrogen limiting growth conditions (67 mg nitrogen per litre at time 0). Batch fermentation of P. putida CA-3 grown on styrene or polystyrene pyrolysis oil in a stirred tank reactor yields PHA at 30% of the cell dry weight (CDW). The feeding of nitrogen at a rate of 1mg N/l/h resulted in a 1.1-fold increase in the percentage of CDW accumulated as PHA. An increase in the rate of nitrogen feeding up to 1.5mg N/l/h resulted in further increases in the percentage of the cell dry weight composed of PHA. However, feeding rates of 1.75 and 2mg N/l/h resulted in dramatic decreases in the percentage of cell dry weight composed of PHA. Interestingly nitrogen was not detectable in the growth medium after 16 h, in any of the growth conditions tested. A higher cell density was observed in cells supplied with nitrogen and thus further increases in the overall production of PHA were observed through nitrogen feeding. The highest yield of PHA was 0.28 g PHA per g styrene supplied with a nitrogen feeding rate of 1.5mg/l/h.     

Effects of Cymbopogon nardus (L.) W. Watson essential oil on the growth and morphogenesis of Aspergillus niger

The growth inhibitory effect of Cymbopogon nardus (L.) W. Watson var. nurdus essential oil on Aspergillus niger (Van Tieghem) mycelium was determined on agar medium. The mycelium growth was completely inhibited at 800 mg/L. This concentration was found to be lethal under the test conditions. Essential oil at 400 mg/L caused growth inhibition of 80% after 4 days of incubation, and a delay in conidiation of 4 days compared with the control. Microscopic observations were carried out to determine the ultrastructural modifications of A. niger hyphae after treatment with C. nardus essential oil. The main change observed by transmission electron microscopy concerned the hyphal diameter and the hyphal wall, which appeared markedly thinner. These modifications in cytological structure might be caused by the interference of the essential oil with the enzymes responsible for wall synthesis which disturb normal growth. Moreover, the essential oil caused plasma membrane disruption and mitochondrial structure disorganization. The findings thus indicate the possibility of exploiting Cymbopogon nardus essential oil as an effective inhibitor of biodegrading and storage-contaminating fungi.     

冠状耳霉产生碱性蛋白酶及其活性*

Thirty isolates of the entomophthotalean fungus, Conidiobolus coronatus, derived fromnaturally infected rice planthoppers were investigated for the achvihes of their alkaline protease.Culturing in sabouraud broth for 48h at 26℃, the isolates yield a dry mycelium biomass of 29.2mg / ml with a maximum of 32mg / ml. The activity of the alkaline protease in supemabot variedgreahy among the isolates, ranging 232-1410u / ml. The specific acihvity was high and venous:11000u / mg for 2 of the isolates tested, 8000-…     

Phytochemical analysis and in vitro evaluation of the biological activity against herpes simplex virus type 1 (HSV-1) of Cedrus libani A. Rich.

Cedrus libani are widely used as traditional medicine in Lebanon for treatment of different infection diseases. In the present study we reported the phytochemical composition analyzed by GC-MS of wood essential oil and cones and leaves ethanol extracts. The main components of wood essential oil were himachalol (22.50%), beta-himachalene (21.90%), and alpha-himachalene (10.50%). Leaves ethanol extract was characterized by a high content of germacrene d (29.40%). The same extract obtained from cones essentially contained alpha-pinene (51.0%) and beta-myrcene (13.0%). Moreover, we investigated extracts, essential oil, and identified compounds for their in vitro antiviral activities against herpes simplex virus type 1 (HSV-1). Cytotoxicity was evaluated by MTT assay in Vero cells. Cones and leaves ethanol extracts exhibited an interesting activity with IC50 of 0.50 and 0.66 mg/ml, respectively, at non-cytotoxic concentration. A comparable activity was found when essential oil was tested (IC50 of 0.44 mg/ml).     

Isolation of Solanapyrones A, B and C from Culture Filture and Spore Germination Fluids of Ascochyta rabiei and Aspects of Phytotoxin Action

Nine isolates of the fungus Ascochyta rabiei have been assayed for their ability to produce solanapyrone toxins. All isolates formed solanapyrone A, B and C which were secreted into the culture medium. Pronounced production of the toxins only occurred after onset of sporulation. The identification of the fungal products was achieved by cochromatography (TLC, HPLC), ¹H-NMR (solanapyrone A and B) and mass spectrometry (solanapyrone B). Work with A. rabiei isolate X showed that cultivation in chickpea seed extract medium in a surface culture provided best conditions for maximal toxin production. The accumulation of solanapyrones over the growth cycle was monitored. Germinating spores produced solanapyrones C and B whereas solanapyrone A was formed from the 6th day of the culture period on. Application of a mixture of solanapyrones A, B and C to leaflets of intact plants from an A. rabiei resistant cultivar (ILC 3279) and a susceptible cultivar (ILC 1929) led to characteristic changes in leaf morphology which had earlier been obsevad in susceptible plants following infection with spores of A. rabiei. Attempts to demonstrate the occurrence of toxins in the infected leaf were unsuccessful. Application of solanapyrones to solanapyrones to chickpea cell suspension cultures (derived from both cultivars) led to pronounced losses in viability and to plasmolysis of cells.     

Potential allelochemicals from the essential oil of Ruta graveolens

The essential oil of aerial parts of Ruta graveolens was obtained by hydrodistillation with a 0.74% yield on a dry weight basis. Thirty-eight components were identified by GC and GC-MS analyses. 2-Ketones predominated in the essential oil, with undecan-2-one (46.8%) and nonan-2-one (18.8%) as the main constituents. The essential oil and some of its constituents were tested for their allelopathic activity in vitro on radish germination and radicle growth in light and darkness. The essential oil and some of its minor constituents were effective and dose-dependent inhibitors of both the germination and radicle growth; 2-ketones are not active. The possible allelopathic activity of rue essential oil and some its isolated constituents is reported.     

Visual micromethod for assay of fungal growth

A visual micromethod for measuring antifungal effects on germination and growth is described. The antifungal agent griseofulvin and the fungus Trichophyton mentagrophytes were used as materials to compare the micromethod with a standard assay based on dry mycelial weight. The micromethod was more sensitive than the weight method in detecting the minimum inhibitory concentration of griseofulvin (0.18 and 35 microgram/ml, respectively). At higher concentrations of griseofulvin (22.5 microgram/ml), the micromethod measured minimal fungal growth that was undetectable on a weight basis. The micromethod showed that griseofulvin does not change the number of spores forming germ tubes. Progressively severe alterations in fungal morphology occured as the concentration of griseofulvin was increased from 0.09 to 22.5 microgram/ml.     

Effect of the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum on the essential oil yield related characters and nutrient acquisition in the crops of different cultivars of menthol mint (Mentha arvensis) under field conditions.

The effects of inoculation with vesicular-arbuscular mycorrhizal (VAM) fungus Glomusfasciculatum on the root colonization, growth, essential oil yield and nutrient acquisition of three cultivars of menthol mint (Mentha arvensis); Kalka, Shivalik and Gomti, were studied under field conditions. The VAM inoculation significantly increased the root colonization, plant height, fresh herbage and dry matter yield. oil content and oil yield as compared to non-inoculated cultivars. The effect of VAM inoculation on the root colonization, growth and yield of mint was more pronounced with the cv Shivalik than the cvs Kalka and Gomati, indicating Shivalik as a highly mycorrhizal dependent genotype. VAM inoculation significantly increased the uptake of N, P and K by shoot tissues of mint, but most markedly increased the uptake of P. The VAM-inoculated mint plants depleted the available N, P and K in the rhizosphere soil as compared to non-inoculated control plants, however the extent of nutrient depletion was greater for P than N and K. We conclude that the VAM inoculation could significantly increase the root colonization, growth, essential oil yield and nutrient acquisition of mint for obtaining economic production under field conditions.     

Effect of calcium on growth performance and essential oil of vetiver grass (Chrysopogon zizanioides) grown on lead contaminated soils

The aim of this study was to investigate effect of calcium on growth, survival, essential oil yield and chemical compositions of vetiver grass grown on lead contaminated soils. Calcium inform of CaCO3 (0, 2000, 4000, 6000 mg Ca kg(-1)) was added to river sand soils containing 4000 mg Pb kg(-1) dry soil. Results showed that, in the absence of calcium treatment, no plants survived after 2 weeks of cultivation, while the rest grew well to the end of the experimental period (42 weeks). Calcium treatments generally resulted in a slight decrease in biomass. Interestingly, an increase in calcium over 2000 mg kg(-1) did not result in a decrease in accumulation of lead in vetiver roots and shoots. The levels of lead in roots and shoots under calcium treatments were around 2000 and 90 mg kg(-1) dry weight, respectively. The addition of CaCO3 did not improve vetiver essential oil yield and chemical composition compared to the control. A level of applied CaCO3 about half of the lead concentration in soils was sufficient to improve vetiver growth and survival, and accumulate high concentrations of lead in the roots. This finding can be applied for re-vegetation of lead contaminated soils using vetiver.