Phytohormone production by three strains of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and possible physiological and technological implications

The aim of this work was to evaluate phytohormone biosynthesis, siderophores production, and phosphate solubilization in three strains (E109, USDA110, and SEMIA5080) of Bradyrhizobium japonicum, most commonly used for inoculation of soybean and nonlegumes in USA, Canada, and South America. Siderophore production and phosphate solubilization were evaluated in selective culture conditions, which had negative results. Indole-3-acetic acid (IAA), gibberellic acid (GA₃), and abscisic acid (ABA) production were analyzed by gas chromatography–mass spectrometry (GC-MS). Ethylene and zeatin biosynthesis were determined by GS–flame ionization detection and high-performance liquid chromatography (HPLC-UV), respectively. IAA, zeatin, and GA₃ were found in all three strains; however, their levels were significantly higher (p < 0.01) in SEMIA5080 (3.8 μg ml⁻¹), USDA110 (2.5 μg ml⁻¹), and E109 (0.87 μg ml⁻¹), respectively. ABA biosynthesis was detected only in USDA110 (0.019 μg ml⁻¹). Ethylene was found in all three strains, with highest production rate (18.1 ng ml⁻¹ h⁻¹) in E109 cultured in yeast extract mannitol medium plus l-methionine. This is the first report of IAA, GA₃, zeatin, ethylene, and ABA production by B. japonicum in pure cultures, using quantitative physicochemical methodology. The three strains have differential capability to produce the five major phytohormones and this fact may have an important technological implication for inoculant formulation.     

Leishmanicidal and antitumoral activities of endophytic fungi associated with the Antarctic angiosperms Deschampsia antarctica Desv. and Colobanthus quitensis (Kunth) Bartl.

A total of 564 isolates of endophytic fungi were recovered from the plants Deschampsia antarctica and Colobanthus quitensis collected from Antarctica. The isolates were screened against parasites Leishmania amazonensis and Trypanosoma cruzi and against the human tumour cell lines. Of the 313 fungal isolates obtained from D. antarctica and 251 from C. quitensis, 25 displayed biological activity. Nineteen extracts displayed leishmanicidal activity, and six inhibited the growth of at least one tumour cell line. These fungi belong to 19 taxa of the genera Alternaria, Antarctomyces, Cadophora, Davidiella, Helgardia, Herpotrichia, Microdochium, Oculimacula, Phaeosphaeria and one unidentified fungus. Extracts of 12 fungal isolates inhibited the proliferation of L. amazonesis at a low IC₅₀ of between 0.2 and 12.5 μg ml⁻¹. The fungus Phaeosphaeria herpotrichoides displayed only leishmanicidal activity with an IC₅₀ of 0.2 μg ml⁻¹, which is equivalent to the inhibitory value of amphotericin B. The extract of Microdochium phragmitis displayed specific cytotoxic activity against the UACC-62 cell line with an IC₅₀ value of 12.5 μg ml⁻¹. Our results indicate that the unique angiosperms living in Antarctica shelter an interesting bioactive fungal community that is able to produce antiprotozoal and antitumoral molecules. These molecules may be used to develop new leishmanicidal and anticancer drugs.     

Antimicrobial activity of crude extracts from mangrove fungal endophytes

The aim of this work was to select endophytic fungi from mangrove plants that produced antimicrobial substances. Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) or minimal fungicidal concentrations (MFC) of crude extracts from 150 isolates were determined against potential human pathogens by a colorimetric microdilution method. Ninety-two isolates (61.3%) produced inhibitory compounds. Most of the extracts (28–32%) inhibited Staphylococcus aureus (MIC/MBC 4–200/64–200 μg ml⁻¹). Only two extracts inhibited Pseudomonas aeruginosa (MIC/MBC 200/>200 μg ml⁻¹). 25.5 and 11.7% inhibited Microsporum gypseum and Cryptococcus neoformans (MIC/MFC 4–200/8–200 μg ml⁻¹ and 8–200/8–200 μg ml⁻¹, respectively), while 7.5% were active against Candida albicans (MIC/MFC 32–200/32–200 μg ml⁻¹). None of the extracts inhibited Escherichia coli. The most active fungal extracts were from six genera, Acremonium, Diaporthe, Hypoxylon, Pestalotiopsis, Phomopsis, and Xylaria as identified using morphological and molecular methods. Phomopsis sp. MA194 (GU592007, GU592018) isolated from Rhizophora apiculata showed the broadest antimicrobial spectrum with low MIC values of 8–32 μg ml⁻¹against Gram-positive bacteria, yeasts and M. gypseum. It was concluded that endophytic fungi from mangrove plants are diverse, many produce compounds with antimicrobial activity and could be suitable sources of new antimicrobial natural products.     

Nutrient regulation of bacterial growth and production of anti-tumor metabolites in <Emphasis Type="Italic">Stigmatella</Emphasis> WXNXJ-B fermentation

The metabolites produced by Stigmatella WXNXJ-B inhibited the growth of tumor cells. The aims of this research were to evaluate the inhibition potency to different tumor cell lines and to study the effects of ammonium, phosphate and iron salts on bacterial growth and production of bioactive metabolites in Stigmatella WXNXJ-B fermentation. The results showed that the chloroform extract (CE-ME) showed the strongest growth inhibition bioactivity on mouse melanoma cell line (B16), murine colon carcinoma cell line (CT-26), human liver carcinoma cell line (HepG2) and human breast cancer cell line (MDA-MB231) in vitro and the IC₅₀ values were 9.94, 7.33, 11.34 and 11.66 μg ml⁻¹ respectively. The IC₅₀ value was above 700 μg ml⁻¹ on normal mouse spleen cells. Morphology happened changes in B16 cells treated with CE-ME. The anti-tumor metabolites were mainly produced during the stationary phase of the bacterial growth. Cell growth was stimulated at the phosphate concentration below 5 mM, but it was inhibited partly with 10 mM phosphate. The production of bioactive substances was inhibited by the phosphate. Ammonium increased the cell growth by 250% at 5 mM addition. The inhibition rate to B16 cells was increased to 89% at the concentration of 40 mM ammonium. The bacteria showed the best growth with 4 mM iron. Iron had little effect on the production at 2 mM, but bigger inhibition effect at higher iron concentration.     

Investigation of the Cytotoxicity of Antimicrobial Peptide P40 on Eukaryotic Cells

The in vitro cytotoxicity of the antimicrobial peptide P40 was investigated. The food grade bacteriocin nisin was also analyzed for comparison. VERO cells were treated with different concentrations (0.02–2.5 μg ml⁻¹) of nisin and P40, and cell viability and plasma membrane integrity were checked by MTT, neutral red uptake (NRU), and lactate dehydrogenase (LDH) assays. In MTT and NRU assays the EC₅₀ to the purified peptide P40 were 0.30 and 0.51 μg ml⁻¹, while values found to nisin were 0.35 and 0.79 μg ml⁻¹, respectively. In the LDH assay, the EC₅₀ was 0.57 and 0.62 μg ml⁻¹ for P40 and nisin, respectively. The peptide P40 revealed higher hemolytical activity (19%) when compared to nisin (4.9%) at the highest concentration tested (2.5 μg ml⁻¹). Relatively few studies about the cytotoxicity of antimicrobial peptides are available. The determination of the cytotoxicity of antimicrobial peptides is an essential step to warrant their safe use.     

Characterization of a cold-tolerant plant growth-promoting bacterium Pantoea dispersa 1A isolated from a sub-alpine soil in the North Western Indian Himalayas

Pantoea dispersa strain 1A is a Gram-negative rod-shaped, yellow-pigmented bacterium isolated on nutrient agar plates incubated at 4°C. The identity of the bacterium was confirmed by sequencing of the 16 S rRNA gene. It was capable of growing at temperatures ranging from 4 to 42°C, but maximum growth was observed at 30°C. It is endowed with multiple plant growth promotion attributes such as phosphate solubilization, IAA production, siderophore production and HCN production, which are expressed differentially at sub-optimal temperatures (15 and 4°C). It was able to solubilize phosphate (17.6 μg of P₂O₅ ml⁻¹ day⁻¹), and produce IAA (3.7 μg ml⁻¹ day⁻¹), at 15°C. Qualitative detection of siderophore production and HCN were also observed at 15°C. At 4°C it was found to express all the plant growth promotion attributes. This bacterial isolate was able to positively influence and promote the growth and nutrient uptake parameters of wheat (cv. VL.802) under glasshouse conditions. Hence in the context, of cold wheat-growing environments, it is proposed that Pantoea dispersa 1A (MTCC 8706), could be deployed as an inoculant to attain the desired results of bacterization.     

Influence of the KDEL signal,DMSO and mannitol on the production of the recombinant antibody 14D9 by long-term <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> cell suspension culture

We have established two transgenic cell suspension culture lines of Nicotiana tabacum that express the catalytic antibody 14D9 as a secretory product (sec-Ab) or as a KDEL-tagged product in the endoplasmic reticulum (Ab-KDEL), respectively. After 3 years of culture, the performance improved to a production level of 0.15 ± 0.03 μg ml⁻¹ on the seventh day of culture for the sec-Ab line and 0.48 ± 0.05 μg ml⁻¹ on the third day for Ab-KDEL line. Analysis of the effect of osmotic stress using mannitol (90 g l⁻¹) as an osmolite revealed that there was a 12-fold increase in antibody yield (1.96 ± 0.20 μg ml⁻¹) on the seventh day of culture in line sec-Ab and a fivefold increase (2.31 ± 0.18 μg ml⁻¹) on the seventh day for line Ab-KDEL. The concentration of the antibody in the culture medium was not significant. Dimethyl sulfoxide used as a permeabilizing agent was not effective in increasing 14D9 yield, but it did cause distinctive cell damage at all concentrations tested.     

Growth retardants stimulate guggulsterone production in the presence of fungal elicitor in fed-batch cultures of <Emphasis Type="Italic">Commiphora wightii</Emphasis>

Guggulsterone, a hypolipidemic natural agent, is produced in resin canals of the plant Commiphora wightii. In this study, the stimulatory effects of growth retardants [ALAR (N,N-dimethylaminosuccinamic acid) and CCC (chlormequat chloride)] and fungal elicitor on guggulsterone accumulation in cell cultures of C. wightii are reported. CCC at 1 mg l⁻¹ enhanced guggulsterone content (~123 μg l⁻¹) when added on the fifth day after inoculation, while ALAR at 2.5 mg l⁻¹ increased guggulsterone content (~116 μg l⁻¹) when added on the tenth day. In a two-stage fed-batch process, combined treatment with fungal elicitor and growth retardant caused a significant increase (~353 μg l⁻¹) in guggulsterone content in cell cultures after 17 days of growth. This represents an approximately fivefold increase over the guggulsterone contents in initial cultures of this plant.     

Plant growth-promoting activity in newly isolated <Emphasis Type="Italic">Bacillus thioparus</Emphasis> (NII-0902) from Western <Emphasis Type="Italic">ghat</Emphasis> forest,India

A Gram-positive rod-shaped bacterium isolated on nutrient agar plates incubated at 28 ± 2°C. The identity of the bacterium was confirmed by sequencing of the 16S rRNA gene and it reveals that it shares highest similarity with Bacillus thioparus CECT 7196^T (99.08%). It was capable of growing at temperatures ranging from 4 to 40°C, but optimum growth was observed at 28 ± 2°C. Strain NII-0902 is endowed with multiple plant growth promotion attributes such as phosphate solubilization, Indole acetic acid (IAA), siderophore and HCN production, which were expressed differentially at sub-optimal temperatures (5–40°C). It was able to solubilize phosphate (17.7 μg ml⁻¹), and produce IAA (139.7 μg ml⁻¹) at 28 ± 2°C. Qualitative detection of siderophore production and HCN were also observed. At 5°C it was found to express all the plant growth promotion attributes except HCN production. The ability to colonize roots is a sine qua non condition for a rhizobacteria to be considered a true plant growth-promoting rhizobacteria (PGPR). Bacillus sp. NII-0902 has a potential ability to colonize roots visualized by transparency, bacterial growth (turbid, milky and narrow zone) along and around roots and truly supported by scanning electron micrograph. Hence, it is proposed that, Bacillus thioparus sp. NII-0902 could be deployed as an inoculant to attain the desired results of bacterization.     

Rhizobacterial potential to alter auxin content and growth of <Emphasis Type="Italic">Vigna radiata</Emphasis> (L.)

Potential of non-symbiotic plant growth promoting rhizobacteria (PGPR) to influence the endogenous indole-3-acetic acid (IAA) content and growth of Vigna radiata (L.) was evaluated. The bacterial strains used belonged to Pseudomonas, Escherichia, Micrococcus and Staphylococcus genera. All strains were able to produce IAA (1.16–8.22 μg ml⁻¹) in the presence of 1,000 μg ml⁻¹ of l-tryptophan as revealed by gas chromatography and mass spectrometric (GC–MS) analysis. However, strains exhibited variable results for other growth promoting traits such as phosphate solubilization and siderophore or hydrogen cyanide production. Bacterial IAA production showed significant positive correlation with endogenous IAA content of roots (r = 0.969; P = 0.01) and leaves (r = 0.905; P = 0.01) under axenic conditions. Bacterization of V. radiata seeds significantly enhanced shoot length (up to 48.10%) and shoot fresh biomass (up to 43.80%) under fully axenic conditions. Bacterial strains applied under wire-house conditions also improved shoot length, number of pods, and grain weight up to 58, 65, and 17.15% respectively, over control. Hence, free living (non-symbiotic) PGPR have the ability to influence endogenous IAA content and growth of leguminous plants.     

Optimization of Antifungal Effect of Surfactin and Iturin to <Emphasis Type="Italic">Penicillium notatum</Emphasis> in Syrup of Peach by RSM

In the paper, the sensitivity of Penicillium notatum to surfactin and iturin was determined, and the optimization of the antifungal of surfactin and iturin to Penicillium notatum in syrup of peach by a response surface methodology (RSM) was researched. Results demonstrated that Penicillium notatum was sensitive to them, whose minimal inhibitory concentration (MIC) was 62.5 and 31.25 μg ml⁻¹ respectively. In the optimization experiment, when the temperature was 2.37°C, the action time was 25.21 h, and the concentration (surfactin/iturin weight ratio 1:1) was 40.26 μg ml⁻¹, Penicillium notatum could be sterilized by 5 orders of magnitude. All the results in the experiment indicated surfactin and iturin could kill remarkably Penicillium notatum in syrup of peach.     

Enhanced regeneration of haploid plantlets from microspores of <Emphasis Type="Italic">Brassica napus</Emphasis> L. using bleomycin,PCIB, and phytohormones

The effects of three periods of incubation (10, 20 and 30 min) at different levels of bleomycin (0, 0.1, 0.2, 0.3, 0.4 and 0.5 μg ml⁻¹), as well as three periods of exposure (12, 24 and 48 h) to different levels of the anti-auxin p-chlorophenoxyisobutyric acid (PCIB), including 1, 2, 3, 4 and 5 mg l⁻¹, on microspore embryogenesis of rapeseed cv. ‘Amica’ were investigated. Microspore embryogenesis was significantly enhanced following 20 min treatment with 0.2 μg ml⁻¹ bleomycin compared with untreated cultures. Highest embryo yield (163 embryos Petri dish⁻¹) was observed with 24 h treatment of 4 mg l⁻¹ PCIB. The highest percentage of secondary embryogenesis was observed on B₅ medium containing 0.15 mg l⁻¹ of gibberellic acid (GA₃) and 0.2 mg l⁻¹ 6-benzyladenine (BA) in 4–6 mm microspore-derived embryos (MDEs). Most callus formed on B₅ medium containing 0.15 mg l⁻¹ GA₃, 0.1 mg l⁻¹ BA and 0.1 mg l⁻¹ indole-3-acetic acid (IAA) when 4–6 mm embryos were used. Regeneration was highest on B₅ medium containing 0.05 mg l⁻¹ GA₃ or 0.1 mg l⁻¹ BA and 0.2 mg l⁻¹ IAA with 2–4 mm embryos. Microspore embryogenesis and plant regeneration could be improved by both bleomycin and PCIB when the appropriate MDE length and phytohormone level were selected.     

Biodegradation of lindane pesticide by non white- rots soil fungus <Emphasis Type="Italic">Fusarium</Emphasis> sp.

Lindane or γ- hexachlorocyclohexane (γ-HCH) is a chlorinated pesticide and its toxic effects on biota necessitate its removal. Microbial degradation is an important process for pesticide bioremediation and the role of soil fungi in recycling of organic matter prompted us to study the biodegradation of lindane using fungi. This study aims at enrichment, isolation and screening of soil fungi capable of metabolizing lindane. Two Fusarium species (F. poae and F. solani) isolated from the pesticide contaminated soil showed better growth on the plates supplemented with lindane as a sole carbon source, when compared with the growth performance of other fungal isolates from the same contaminated soil. However, ANOVA revealed a significant difference in fungal biomass production in both F. poae (F = 22.02; N = 15; P < 0.001) and F. solani (F = 268.75; N = 15; P < 0.001) across different lindane concentrations (0–600 μg ml⁻¹). Growth of both Fusarium sp. was maximum at a lindane concentration of 100 μg ml⁻¹, while minimum at 600 μg ml⁻¹ concentrations. Results on the time dependent release of chlorine by the Fusarium strains in the presence of various concentration of lindane showed the highest mineralization of the pesticide on 10th day of incubation. Time dependent variations in the release of chlorine from 1st to 10th day by both the selected fungal strains were found to be statistically significant. A significant positive relationship exists between fungal biomass increase and chlorine release existed for both F. solani (R2 = 0.960) and F. poae (R2 = 0.628). The results of gas chromatograph analysis of γ- HCH confirmed the biodegradation and utilization of γ- HCH by F. poae and F. solani. The data on lindane degradation by the two fungal strains demonstrated that the biodegradation of lindane by F. solani (59.4%) was slightly higher than that by the F. poae (56.7%).     

Assessment of antifungal effects of a novel compound from <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> against <Emphasis Type="Italic">Fusarium solani</Emphasis> by fluorescent staining

A novel compound CF66I produced by Burkholeria cepacia was investigated for its antifungal effects against Fusarium solani by three different fluorescent dyes. Dual staining with propidium iodide (PI) and fluorescein diacetate (FDA) demonstrated high doses of CF66I (120.0 μg ml⁻¹) killed the fungi by acting primarily on the cell membrane. However, at fungistatic concentration (20.0 μg ml⁻¹) of this compound, microscopic observations revealed swelling hyphae with abnormal chitin deposition, as determined by Calcofluor white (CFW) staining, which was indicative of the alterations in cell wall structure. In addition, inhibition of intracellular esterases activity was observed. These results led us to conclude that low doses of CF66I probably inhibited the fungal growth by interfering with the cell metabolic pathways.     

Plant-growth-promoting compounds produced by two agronomically important strains of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>, and implications for inoculant formulation

We evaluated phytohormone and polyamine biosynthesis, siderophore production, and phosphate solubilization in two strains (Cd and Az39) of Azospirillum brasilense used for inoculant formulation in Argentina during the last 20 years. Siderophore production and phosphate solubilization were evaluated in a chemically defined medium, with negative results. Indole 3-acetic acid (IAA), gibberellic acid (GA₃), and abscisic acid (ABA) production were analyzed by gas chromatography-mass spectrometry. Ethylene, polyamine, and zeatin (Z) biosynthesis were determined by gas chromatography-flame ionization detector and high performance liquid chromatography (HPLC-fluorescence and -UV), respectively. Phytohormones IAA, Z, GA₃, ABA, ethylene, and growth regulators putrescine, spermine, spermidine, and cadaverine (CAD) were found in culture supernatant of both strains. IAA, Z, and GA₃ were found in all two strains; however, their levels were significantly higher (p < 0.01) in Cd (10.8, 2.32, 0.66 μg ml⁻¹). ABA biosynthesis was significantly higher (p < 0.01) in Az39 (0.077 μg ml⁻¹). Ethylene and polyamine CAD were found in all two strains, with highest production in Cd cultured in NFb plus l-methionine (3.94 ng ml⁻¹ h⁻¹) and Az39 cultured in NFb plus l-lysine (36.55 ng ml⁻¹ h⁻¹). This is the first report on the evaluation of important bioactive molecules in strains of A. brasilense as potentially capable of direct plant growth promotion or agronomic yield increase. Az39 and Cd showed differential capability to produce the five major phytohormones and CAD in chemically defined medium. This fact has important technological implications for inoculant formulation as different concentrations of growth regulators are produced by different strains or culture conditions.     

Isolation of antifouling compounds from the marine bacterium, <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> SCH0402

Two compounds, 2-hydroxymyristic acid (HMA) and cis-9-oleic acid (COA), were isolated from a chloroform extract of the marine bacterium, Shewanella oneidensis SCH0402. In a spectrophotometer-based chemotaxis assay, HMA completely eliminated the optical density (OD) of Alteromonas marina SCH0401 and Bacillus atrophaeus SCH0408, motile, fouling bacteria, at 100 and 1000 μg ml⁻¹, respectively. COA similarly decreased the OD of A. marina and B. atrophaeus by 100% at 1000 μg ml⁻¹. The commercially available, highly toxic anti-fouling compound, tributyltin oxide (TBTO) never reduced the OD of the target bacteria by 100% even at higher concentration. Instead, all the test bacterial cells were killed at higher than 1000 μg ml⁻¹ of concentration. Both HMA and COA inhibited germination of Ulva pertusa spores completely at 10 and 100 μg ml⁻¹, respectively, while TBTO inhibited germination at 0.01 μg ml⁻¹. However, in field assays, both HMA and COA showed anti-fouling activities as potent as TBTO against a wide range of fouling organisms, including micro- and macro-algae, barnacles, and mussels. The average fouling coverage on the surface of the control panel was 93 ± 6% after 1.5 years but no fouling was observed on the surface of the test panel onto which each compound was applied separately. Thus, bacterial repellent compounds can be used as substitutes for potent toxic anti-fouling compounds, resulting in higher standards of environmental safety without loss of anti-fouling performance.     

Marine Bacterium Derived Lipopeptides: Characterization and Cytotoxic Activity Against Cancer Cell Lines

A marine Bacillus circulans DMS-2 was able to grow and produce biosurfactant on glucose mineral salts medium (GMSM) with a reduction in the surface tension up to 27 mN m⁻¹. The microorganism produced 1.64 ± 0.1 g l⁻¹ of crude biosurfactant. The lipopeptide nature of the produced biosurfactant was confirmed by primulin and ninhydrin assays using High Performance Thin Layer Chromatography (HPTLC). Preparative thin layer chromatography (TLC) was performed to purify the lipopeptides from the crude biosurfactant. The critical micelle concentrations (CMC) of the crude and purified products were found to be 90 and 40 mg l⁻¹ respectively. Fourier transform infrared spectrophotometer (FTIR) and matrix assisted laser desorption/ionization time of flight (MALDI-ToF) mass spectral analysis revealed the identity of the produced lipopeptides as surfactin (m/z 1,023 Da) and fengycin (m/z 1,495 Da) isoforms. The purified marine lipopeptides displayed a significant antiproliferative activity against the human colon cancer cell lines HCT-15 (IC₅₀ 80 μg ml⁻¹) and HT-29 (IC₅₀ 120 μg ml⁻¹).     

Changes in bacterial and ciliate densities with trophic status in Mediterranean shallow lakes

Ciliate and bacterial densities and their link with eutrophication were studied in fourteen shallow lakes in northwest Spain. Total phosphorus (TP) in these lakes varied between 30 μg l⁻¹ and 925 μg l⁻¹ and chlorophyll a concentration (chla) between 0.5 μg l⁻¹ and 107 μg l⁻¹. Bacterial abundance ranged from 1 × 10⁶ to 14 × 10⁶ cells ml⁻¹, while ciliate abundance ranged from 0.6 cells ml⁻¹ to 229 cells ml⁻¹. Lakes were classified into three trophic types from their TP and chla concentrations. Bacterial abundance was significantly correlated with trophic type, as well as with TP and with chla separately, whereas ciliate abundance was only correlated with chla. No significant relationship could be established between bacterial and ciliate abundance across the trophic gradient. A general pattern was observed in the ratios of bacterial abundance to TP and chla concentrations, of decreasing ratios with increases in the nutrient loading. This pattern was not found for ciliates. The dominant zooplankton group in 13 of the 14 lakes studied was Rotifera, which accounted for a mean of 71% of total zooplankton abundance (41% of zooplankton biomass). The positive correlation between bacteria and ciliates with this group, and the absence of any relationship with Cladocera suggest that top down control by cladocerans was weaker in our lakes than previously shown in northern European shallow lakes. Rotifers could be important predators of bacteria in the high-nutrient lakes of our study. Higher slopes of regressions on bacterial abundance towards the hypertrophic range indicate that top-down control was weaker in our lakes than in northern European shallow lakes.     

In vitro efficacy of <Emphasis Type="Italic">Hyptis suaveolens</Emphasis> L. (Poit.) essential oil on growth and morphogenesis of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">gladioli</Emphasis> (Massey) Snyder &; Hansen

Hyptis suaveolens L. (Poit.) essential oil was tested in vitro on the growth and morphogenesis of Fusarium oxysporum f.sp. gladioli (Massey) Snyder & Hansen, which causes Fusarium corm rot and yellows in various susceptible cultivars of gladiolus. The fungitoxicity of the oil was measured by percentage radial growth inhibition using the poisoned food technique (PF) and volatile activity assay (VA). The mycelial growth of the test fungus was completely inhibited at 0.998 and 0.748 μg ml⁻¹ concentration of oil in PF and VA, respectively. Essential oil was found to be fungicidal in nature at 1.247 and 0.998 μg ml⁻¹ concentration of oil in PF and VA, respectively. Determination of conidial germination in the presence of oil was also carried out and it was found that the oil exhibited 100% inhibition of conidial germination at 0.450 μg ml⁻¹ concentration. The effect of essential oil on the yield of mycelial weight was observed and it was found that at 0.873 μg ml⁻¹ concentration no mycelium was recorded and 100% inhibition was observed. The fungitoxicity of oil did not change even on exposure to 100°C temperature or to autoclaving, and the oil also retained its fungicidal nature even after storage of 24 months. The main changes observed under light microscopy after oil treatment were a decrease and loss of conidiation and anomalies in the hyphae such as a decrease in the diameter of hyphae and granulation of cytoplasm. The treatment of the oil also showed highly reduced cytoplasm in the hyphae, showing clear retraction of the cytoplasm from the hyphae and ultimately in some areas hyphae without cytoplasm were also found. GC-MS studies of the essential oil revealed that the oil consisted of 24 compounds with 1,8-cineole as major component accounting for 44.4% of the total constituents.     

Synergistic effect of green tea extract and probiotics on the pathogenic bacteria, <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis>

The aim of this study was to assess the effect of a commercial green tea extract (TEAVIGO™) on the microbial growth of three probiotic strains (Lactobacillus and Bifidobacterium), as well as three pathogenic bacteria. MIC and co-culture studies were performed. The MICs of the green tea extract against Staphylococcus aureus and Streptococcus pyogenes (100 μg ml⁻¹) were considerably lower than those against the probiotic strains tested (>800 μg ml⁻¹) and Escherichia coli (800 μg ml⁻¹). In co-culture studies, a synergistic effect of the probiotic strains and the green tea extract was observed against both Staph. aureus and Strep. pyogenes. Green tea extract in combination with probiotics significantly reduced the viable count of both pathogens at 4 h and by 24 h had completely abolished the recovery of viable Staph. aureus and Strep. pyogenes. These reductions were more significant than the reductions induced by probiotics or green tea extracts used separately. These results demonstrate the potential for combined therapy using the green tea extract plus probiotics on microbial infections caused by Staph. aureus and Strep. pyogenes. As probiotics and the green tea extract are derived from natural products, treatment with these agents may represent important adjuncts to, or alternatives to, conventional antibiotic therapy.