Effects of water-level fluctuations on the arbuscular mycorrhizal colonization of Typha latifolia L.

Flooding has been described as one of the primary factors affecting arbuscular mycorrhizal (AM) colonization in wetlands. We investigated the effect of water-level fluctuations on AM colonization of Typha latifolia L. using an experimental wetland in southeastern Idaho, USA that received intermittent flows. Unlike previous research that has examined the effect of flooding on AM fungi using topographic gradients, we replicated flooding in time by sampling across multiple flooding events. AM colonization of T. latifolia occurred during flooded and unflooded periods, but was markedly reduced at drawdown. Both hyphal (R = 0.74, P = 0.015) and arbuscular (R = 0.67, P = 0.033) colonization were positively correlated with the length of the unflooded period. Taken together, the length of the unflooded period and soil moisture explained 83% of the variation in mean hyphal colonization (R² = 0.83, P = 0.001). Overall, the results of this investigation show that drawdown represents a period of reduced AM colonization in T. latifolia.     

Differential effects of fenpropimorph and fenhexamid, two sterol biosynthesis inhibitor fungicides, on arbuscular mycorrhizal development and sterol metabolism in carrot roots

Sterols composition of transformed carrot roots incubated in presence of increasing concentrations of fenpropimorph (0.02; 0.2; 2 mg l⁻¹) and fenhexamid (0.02; 0.2; 2; 20 mg l⁻¹), colonized or not by Glomus intraradices was determined. In mycorrhizal roots treated with fenpropimorph, normal Δ⁵-sterols were replaced by unusual compounds such as 9β,19-cyclopropylsterols (24-methylpollinastanol), Δ^8,14-sterols (ergosta-8,14-dienol, stigmasta-8,14-dienol), Δ⁸-sterols (Δ⁸ sitosterol) and Δ⁷-sterols (ergosta-7,22-dienol). After application of fenpropimorph, a drastic reduction of the mycorrhizal root growth, root colonization and extraradical fungal development was observed. Application of fenhexamid did not modify sterol profiles and the total colonization of roots. But the arbuscule frequency of the fungal partner was significantly affected.Comparison of the effects caused by the tested fungicides indicates that the usual phytosterols may be involved in symbiosis development. Indeed, observed modifications of root sterols composition could explain the high fenpropimorph toxicity to the AM symbiosis. However, the absence of sterolic modifications in the roots treated with fenhexamid could account for its more limited impact on mycorrhization.     

ACC deaminase-containing Arthrobacter protophormiae induces NaCl stress tolerance through reduced ACC oxidase activity and ethylene production resulting in improved nodulation and mycorrhization in Pisum sativum

Induction of stress ethylene production in the plant system is one of the consequences of salt stress which apart from being toxic to the plant also inhibits mycorrhizal colonization and rhizobial nodulation by oxidative damage. Tolerance to salinity in pea plants was assessed by reducing stress ethylene levels through ACC deaminase-containing rhizobacteria Arthrobacter protophormiae (SA3) and promoting plant growth through improved colonization of beneficial microbes like Rhizobium leguminosarum (R) and Glomus mosseae (G). The experiment comprised of treatments with combinations of SA3, G, and R under varying levels of salinity. The drop in plant biomass associated with salinity stress was significantly lesser in SA3 treated plants compared to non-treated plants. The triple interaction of SA3 + G + R performed synergistically to induce protective mechanism against salt stress and showed a new perspective of plant-microorganism interaction. This tripartite collaboration increased plant weight by 53%, reduced proline content, lipid peroxidation and increased pigment content under 200 mM salt condition. We detected that decreased ACC oxidase (ACO) activity induced by SA3 and reduced ACC synthase (ACS) activity in AMF (an observation not reported earlier as per our knowledge) inoculated plants simultaneously reduced the ACC content by 60% (responsible for generation of stress ethylene) in SA3 + G + R treated plants as compared to uninoculated control plants under 200 mM salt treatment. The results indicated that ACC deaminase-containing SA3 brought a putative protection mechanism (decrease in ACC content) under salt stress, apart from alleviating ethylene-induced damage, by enhancing nodulation and AMF colonization in the plants resulting in improved nutrient uptake and plant growth.     

Effect of a formulation of Bacillus firmus on root-knot nematode Meloidogyne incognita infestation and the growth of tomato plants in the greenhouse and nursery

Bacillus firmus, commercial WP formulation (BioNem) was evaluated against the root-knot nematode Meloidogyne incognita in a laboratory, greenhouse and under field conditions on tomato plants. In the laboratory tests, an aqueous suspension of BioNem at 0.5%, 1%, 1.5% and 2% concentration reduced egg hatching from 98% to 100%, 24-days after treatment. Treatment of second-stage juveniles with 2.5% and 3% concentration of BioNem, caused 100% inhibition of mobility, 24 h after treatment. In the green house trials, BioNem applied at 8 g/pot (1200 cc soil) planted with a tomato seedlings reduced gall formation by 91%, final nematode populations by 76% and the number of eggs by 45%. Consequently, plant height and biomass was increased by 71% and 50%, respectively, compared to the untreated control, 50-days after treatment application. Application of BioNem at 16 g/pot was phytotoxic to plants. In the field trails, BioNem applied at 200 and 400 kg ha⁻¹ was effective in reducing the number of galls (75-84%), and increased shoot height (29-31%) and weight (20-24%) over the untreated control, 45-days after treatment. Our results indicate that B. firmus is a promising microorganism for the biological control of M. incognita in tomato pots.     

Carnivory in plants as a beneficial trait in wetlands

A new hypothesis for the benefit of carnivory in plants (i.e., an alternative to aerenchyma for avoiding hypoxia) is evaluated. Root porosity and root depth were quantified in eight carnivorous plant species and 48 non-carnivorous species within a nutrient-poor wet pine savanna in south Mississippi, USA. Carnivorous and non-carnivorous plant species were contrasted with respect to their indication of wetlands, open habitats, and habitats with nutrient-poor soils. We used path analysis, multiplicative regression, and a field experiment to test hypotheses of the effects of soil moisture/hypoxia on the abundance of carnivorous and non-carnivorous plants. All carnivorous plant species produced non-porous roots (or no roots), which were shallower than the average for non-carnivorous plants (6.9 ± 0.95 cm vs. 11.9 ± 0.96 cm), even after correcting for plant size. Root porosity in non-carnivorous species (mean = 22%) was positively correlated with root depth (r = 0.6). Despite lacking porous roots, carnivorous plants were four times more indicative of wetland habitats than were the non-carnivorous species encountered in the wetland studied here. Carnivorous plants, along with non-carnivorous plants with well-developed aerenchyma, were positively associated with the wettest microsites and were more negatively affected by elevating the substrate than were non-carnivorous plants with low-porosity roots. Non-carnivorous plants with shallow roots, while less indicative of wetlands and less abundant in wet microsites of the wet pine savanna than were carnivorous plants, were no less indicative of nutrient-poor soils than were carnivorous plants. Results supported the hypothesis that carnivory is advantageous in wet soils and disadvantageous in drier (including mesic) soils and are more indicative of wetland conditions than of low soil fertility.     

Inoculation of Rhizobium (VR-1 and VA-1) induces an increasing growth and metal accumulation potential in Vigna radiata and Vigna angularis L. growing under fly-ash

Fly-ash-tolerant Rhizobium strains were isolated from plants grown in fly-ash-contaminated soil, axenically under laboratory conditions. Saplings of both plants were raised in N₂-free Jenson medium and inoculated with 2.6 × 10⁸ cell ml⁻¹ and 5.2 × 10⁸ cell ml⁻¹ of culture after 10 d of growth. Plants were transferred into 100% fly-ash under natural condition. Rhizobium-inoculated plants grown on 100% fly-ash showed marked increase in relation to root-shoot length, biomass yield, photosynthetic pigment, protein content and nodulation frequency compared to uninoculated plant grown in control (100% fly-ash). Inoculation of fly-ash-tolerant Rhizobium increased the accumulation of Fe, Zn, Cu Cd and Cr in different tissues vis-à-vis enhanced translocation of metals to the aboveground part of plant. Although inoculation of fly-ash-tolerant Rhizobium strains (VR-1 and VA-1) enhanced the translocation of more Fe to shoot parts, nevertheless, the amount of Rhizobium inoculants supplied to the plant was found to be very important since it has a positive role in increasing plant growth through increased N₂ supply via nitrogenase activity. Results suggest that an integrated approach employing biotechnological means and inoculation of plants with host-specific fly-ash-tolerant Rhizobium strain may prove a stimulus to a fly-ash management programme.     

A multicenter pilot external quality assessment programme to assess the quality of molecular detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae

An external quality assessment (EQA) panel consisting of a total of 13 samples in broncho alveolar lavage (BAL) or transport medium was prepared to assess the proficiency of laboratories in the correct detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by nucleic acid amplification techniques (NAATs) (6 samples containing various concentrations (4.9-490 inclusion forming units (IFU)/ml) of C. pneumoniae, 5 samples containing various concentrations (20-5000 color-changing units (CCU)/ml) of M. pneumoniae and 2 samples negative for both).Seventy-nine laboratories from 18 countries participated in this EQA study. Sixty-four datasets were returned for C. pneumoniae (n = 5 conventional commercial, n = 10 conventional in-house, n = 4 real-time commercial, n = 43 real-time in-house, and n = 2 SDA). Sixty-seven datasets were obtained for M. pneumoniae (n = 5 conventional commercial, n = 10 conventional in-house, n = 4 real-time commercial, n = 46 real-time in-house, and n = 2 strand displacement amplification (SDA)). For the total panels, correct results per sample varied between 95.3% and 100% for C. pneumoniae and between 53.7% and 95.5% for M. pneumoniae. In general, commercial conventional NAATs showed possible sensitivity issues when compared to conventional in-house NAATs for both organisms. On the other hand, real-time commercial NAATs scored better than real-time in-house assays in terms of sensitivity for both organisms. For C. pneumoniae and M. pneumoniae, 0.8% and 2.2% true false-positive results and 1.9% and 2.0% false positives were reported in the samples spiked with the other organism.Analysis of the data for C. pneumoniae showed that the concentrations used were easily detectable by the vast majority of participants. The percentage of correct qualitative results for M. pneumoniae demonstrated that the concentrations included in this panel proved challenging for a number of participants.     

Alleviation of drought stress of marigold (Tagetes erecta) plants by using arbuscular mycorrhizal fungi

The effect of an arbuscular mycorrhizal fungus “AMF” (Glomus constrictum Trappe) on growth, pigments, and phosphorous content of marigold (Tagetes erecta) plant grown under different levels of drought stress was investigated. The applied drought stress levels reduced growth vigor (i.e. plant height, shoot dry weight, flower diameter as well as its fresh and dry weights) of mycorrhizal and non-mycorrhizal plant as compared to control plant (non-drought stressed plant). The presence of mycorrhizal fungus, however, stimulated all growth parameters of the treated plant comparing to non-mycorrhizal treated plant. The photosynthetic pigments (carotene in flowers and chlorophylls a and b in leaves) were also stimulated by the mycorrhizal fungi of well-watered as well as of water-stressed plants. The total pigments of mycorrhizal plants grown under well-watered conditions were higher than those of non-mycorrhizal ones by 60%. In most cases, drought-stressed mycorrhizal plants were significantly better than those of the non-mycorrhizal plants. So, the overall results suggest that mycorrhizal fungal colonization affects host plant positively on growth, pigments, and phosphorous content, flower quality and thereby alleviates the stress imposed by water with holding.     

Development of cesium phosphotungstate salt and chitosan composite membrane for direct methanol fuel cells

A novel composite membrane has been developed by doping cesium phosphotungstate salt (Cs_xH_3−xPW₁₂O₄₀ (0 ≤ x ≤3), Cs_x-PTA) into chitosan (CTS/Cs_x-PTA) for application in direct methanol fuel cells (DMFCs). Uniform distribution of Cs_x-PTA nanoparticles has been achieved in the chitosan matrix. The proton conductivity of the composite membrane is significantly affected by the Cs_x-PTA content in the composite membrane as well as the Cs substitution in PTA. The highest proton conductivity for the CTS/Cs_x-PTA membranes was obtained with x = 2 and Cs₂-PTA content of 5 wt%. The value is 6 × 10⁻³ S cm⁻¹ and 1.75 × 10⁻² S cm⁻¹ at 298 K and 353 K, respectively. The methanol permeability of CTS/Cs₂-PTA membrane is about 5.6 × 10⁻⁷, 90% lower than that of Nafion-212 membrane. The highest selectivity factor (φ) was obtained on CTS/Cs₂-PTA-5 wt% composite membrane, 1.1 × 10⁴/S cm⁻³ s. The present study indicates the promising potential of CTS/Cs_x-PTA composite membrane as alternative proton exchange membranes in direct methanol fuel cells.     

Differential sensitivity of four Lemnaceae species to zinc sulphate

Lemnaceae are currently the only freshwater plants required for regulatory toxicity testing of pesticides and other chemicals. Toxicological protocols allow for the use of different Lemnaceae species in tests. However, few studies have compared the relative sensitivity of individual duckweed species. Zinc is an essential plant nutrient but is also a common pollutant in aquatic environments and elevated levels are phytotoxic. This study shows that four species of Lemnaceae differ in their relative sensitivities to zinc sulphate, a commonly used reference chemical. Comparative zinc sensitivity, in order, from most tolerant to most sensitive was: Landoltia punctata > Lemna minor > Wolffia brasiliensis > Lemna gibba. Zinc sensitivity was also endpoint dependant. EC₅₀ values typically increased in order of: specific biomass growth rate < specific frond number growth rate < chlorophyll absorbance. However, specific frond number growth rate was the most sensitive endpoint for L. punctata. Unlike the other species, L. punctata displayed no significant colony disintegration. Lemna species and L. punctata appear to be employing distinct response strategies when exposed to zinc. L. gibba and L. minor produce and release young, single fronds which are severely affected by zinc. In contrast, L. punctata produces fewer fronds, which are not released and form large colonies of high biomass that are relatively zinc tolerant.     

Honeybush (Cyclopia spp.): From local cottage industry to global markets — The catalytic and supporting role of research

Honeybush tea (Cyclopia spp.), one of the traditional South African herbal teas with a long history of regional use, remained a cottage industry until the mid-1990s when researchers were instrumental in the development of a formal agricultural and agro-processing industry. It is one of the few indigenous South African plants that made the transition from the wild to a commercial product during the past 100 years. Research activities during the past 20 years included propagation, production, genetic improvement, processing, composition and the potential for value-adding. The present review provides an up-to-date and comprehensive record of the development of the South African honeybush industry, against the background of the historical highlights in the making of an industry. It provides a blueprint of the processes and actions involved in the development of a new agricultural and agro-processing industry from an herbal plant. Insight into challenges faced by the industry and future research needed to keep it competitive are provided.     

Differential effect of sample preservation methods on plant and arbuscular mycorrhizal fungal DNA

A wide range of methods are commonly used for preserving environmental samples prior to molecular analyses. However, the effect of these preservation methods on fungal DNA is not understood. The objective of this study was to test the effect of eight different preservation methods on the quality and yield of DNA extracted from Bromus inermis and Daucus carota roots colonized by the arbuscular mycorrhizal (AM) fungus, Glomus intraradices. The total DNA concentration in sample extracts was quantified using spectrophotometry. Samples that were frozen (− 80 ºC and − 20 ºC), stored in 95% ethanol, or silica gel dried yielded total (plant and fungal) DNA concentrations that were not significantly different from fresh samples. In contrast, samples stored in CTAB solution or freeze-dried resulted in significantly reduced DNA concentrations compared with fresh samples. The preservation methods had no effect on the purity of the sample extracts for both plant species. However, the DNA of the dried samples (silica gel dried, freeze-dried, heat dried) appeared to be slightly more degraded compared with samples that remained hydrated (frozen, stored in ethanol or CTAB solutions) during storage when visualized on a gel. The concentration of AM fungal DNA in sample extracts was quantified using TaqMan real time PCR. Methods that preserved samples in hydrated form had similar AM fungal DNA concentrations as fresh samples, except D. carota samples stored in ethanol. In contrast, preservation methods that involved drying the samples had very low concentrations of AM fungal DNA for B. inermis, and nearly undetectable for D. carota samples. The drying process appears to be a major factor in the degradation of AM fungal DNA while having less of an impact on plant DNA. Based on these results, samples that need to be preserved prior to molecular analysis of AM fungi should be kept frozen to minimize the degradation of plant and AM fungal DNA.     

Effect of biosurfactant and fertilizer on biodegradation of crude oil by marine isolates of Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa

This study was conducted to investigate the effects of fertilizers and biosurfactants on biodegradation of crude oil by three marine bacterial isolates; Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa. Five sets of experiments were carried out in shake flask and microcosm conditions with crude oil as follows: Set 1-only bacterial cells added (no fertilizer and biosurfactant), Set 2-with additional fertilizer only, Set 3-with additional biosurfactant only, Set 4-with added biosurfactant + fertilizer, Set 5-with no bacterial cells added (control), all the above experimental sets were incubated for 168 h. The biosurfactant + fertilizer added Set 4, resulted in maximum crude oil degradation within shake flask and microcosm conditions. Among the three bacterial isolates, P. aeruginosa and biosurfactant produced by this strain resulted in maximum crude oil degradation compared to the other two bacterial strains investigated. Interestingly, when biosurfactant and bacterial cells were used (Set 3), significant oil biodegradation activity occurred and the difference between this treatment and that in Set 4 with added fertilizer + biosurfactant were only 4-5% higher degradation level in shake flask and 3.2-7% in microcosm experiments for all three bacterial strains used. It is concluded that, biosurfactants alone capable of promoting biodegradation to a large extent without added fertilizers, which will reduce the cost of bioremediation process and minimizes the dilution or wash away problems encountered when water soluble fertilizers used during bioremediation of aquatic environments.     

Inhibitory effect of essential oils of Allium sativum and Piper longum on spontaneous muscular activity of liver fluke, Fasciola gigantica

Effects of essential oil of Allium sativum (garlic) and Piper longum (Indian long pepper) were evaluated on muscular activity of whole Fasciola gigantica and its strip preparation. The whole flukes and longitudinal strip preparations of the flukes were isometrically mounted to record the spontaneous muscular activity (SMA) and to evaluate effects of cumulative doses (0.1, 0.3, 1.0 and 3.0 mg/ml) of the plant essential oils. Whole flukes and the strip preparations exhibited continuous SMA without any significant difference in its baseline tension, frequency and amplitude for 2 h. Essential oil of A. sativum produced significant reduction in the frequency and the amplitude of the SMA of whole fluke at 1 and 3 mg/ml concentrations. It caused complete paralysis of the fluke after 15 min of administration of 3 mg/ml concentration. Similar to whole fluke, essential oil of A. sativum (3 mg/ml) also produced flaccid paralysis in the strip preparations of the flukes. Essential oil of P. longum firstly induced marked excitatory effect and then there was flaccid paralysis of the whole fluke following 15 min exposure at 3 mg/ml concentration. Complete flaccid paralysis of the strip preparation was also ensued after 15 min of administration of 3 mg/ml concentration of P. longum. In both the essential oils, the whole fluke and strip preparations did not recover from paralysis following 2-3 washes. In conclusion, the observations demonstrated irreversible paralytic effect of essential oils of A. sativum and P. longum on F. giganticain vitro which might possibly help to developing herbal-based anthelmintic.     

Comparative growth kinetics and virulence of four different isolates of entomopathogenic fungi in the house fly (Muscadomestica L.)

Virulence (speed of kill) of a fungal entomopathogen against a particular host insect depends on biological properties of the specific isolate-host combination, together with factors such as fungal dose. How these intrinsic and extrinsic factors affect the actual pattern and extent of fungal growth invivo is poorly understood. In this study we exposed adult house flies (Muscadomestica L.) to surfaces treated with high and low doses of Beauveriabassiana (isolates BbGHA and Bb5344), Metarhiziumanisopliae (strain MaF52) and M.anisopliae var. acridum (isolate Ma189) and used quantitative real-time PCR with species-specific primers to examine the relationship between fungal growth kinetics and virulence. At the highest dose, all fungal isolates killed flies significantly faster than controls, with BbGHA, Bb5344 and MaF52 roughly equivalent in virulence (median survival time (±SE) = 5.0 ± 0.10, 5.0 ± 0.08 and 5.0 ± 0.12 days, respectively) and Ma189 killing more slowly (MST = 8.0 ± 0.20 days). At the lower dose, effective virulence was reduced and only flies exposed to isolates BbGHA and Bb5344 died significantly faster than controls (MST = 12 ± 1.36, 15 ± 0.64, 18 ± 0.86 and 21.0 ± 0.0 days for BbGHA, Bb5344, MaF52 and Ma189, respectively). Real-time PCR assays revealed that flies exposed to surfaces treated with the high dose of spores had greater spore pickup than flies exposed to the low dose for each isolate. After pickup, a general pattern emerged for all isolates in which there was a significant reduction of recovered fungal DNA 48 h after exposure followed by a brief recovery phase, a stable period of little net change in fungal sequence counts, and then a dramatic increase in sequence counts of up to three orders of magnitude around the time of host death. However, while the patterns of growth were similar, there were quantitative differences such that higher final sequence counts were recovered in insects infected with the most lethal isolates and with the higher dose. These results suggest that variation in virulence between isolates, species and doses is determined more by quantitative rather than qualitative differences in fungal growth kinetics.     

Virulence of Hypocreales fungi to pecan aphids (Hemiptera: Aphididae) in the laboratory

There is need for efficacious biocontrol agents for aphids in commercial orchards. As a preliminary step to this end we determined the virulence of several Hypocreales fungi to pecan aphids. In the first experiment we tested the virulence of Isaria fumosorosea (ARSEF 3581) blastospores to three pecan aphids Monellia caryella, Melanocallis caryaefoliae, and Monelliopsis pecanis under laboratory conditions. Rates of 1 × 10⁷ or 1 × 10⁸ spores per ml were applied in 2 ml via a spray tower to 90 mm Petri dishes containing 10 aphids each. Mortality and mycosis were determined after 24, 48 and 72 h. Treatment effects were observed by 48 h post-application, and by 72 h the higher application rate caused >90% mortality and mycosis in M. caryella and M. caryaefoliae, whereas <70% was observed in M. pecanis.We conducted two subsequent experiments (Experiments 2 and 3), using the same methodology, to compare the virulence of several Hypocreales species and strains against the aphid of primary economic concern to most pecan growers, M. caryaefoliae. In Experiment 2, we compared blastospores and conidia of two I. fumosorosea strains (ARSEF 3581 and ATCC 20874 [= strain 97]). The blastospores of ARSEF 3581 and conidia of ATCC 20874 showed higher virulence than other treatments and thus were included in Experiment 3, which also compared the virulence of conidia of Beauveria bassiana (GHA strain) and Metarhizium anisopliae (F52 strain). Results in Experiment 3 indicated the highest virulence in I. fumosorosea 3581 blastospores and M. anisopliae (F52) followed by I. fumosorosea (20874) conidia. The detection of pathogenicity to pecan aphids establishes the potential for commercial usage and additional study. Results reported here will narrow treatments to test in future greenhouse and field trials.     

Antimicrobial activity and in vitro cytotoxicity of selected South African Helichrysum species

The antimicrobial activity of 35 indigenous South African Helichrysum species was determined against six microorganisms. Seven of the 36 chloroform:methanol (1:1) extracts (leaf and stem extracts for all plants and an additional flower extract for H. rugulosum) exhibited minimum inhibitory concentration (MIC) values lower than 0.1 mg/ml against Bacillus cereus and/or Staphylococcus aureus. The in vitro cytotoxicity [against transformed human kidney epithelial (Graham) cells, MCF-7 breast adenocarcinoma and SF-268 glioblastoma cells] of these extracts was also determined at a concentration of 0.1 mg/ml using the sulforhodamine B (SRB) assay. For seven species less than 25% growth was observed for the Graham and MCF-7 cell lines at the test concentration.     

Interaction of Pratylenchus brachyurus and Gigaspora margarita on Cotton

An endomycorrhizal fungus, Gigaspora margarita, was more effective in stimulating the growth of cotton (Gossypium hirsutum) ''Coker 201'' at a low fertility level (1.77 gm 10-10-10 N-P-K/pot) than doubling the fertility rate for nonmycorrhizal plants. Gigaspora margarita alone stimulated shoot growth (height, weight, and flower production by 96%, 553%, and 760%, respectively) and root growth (385%) over that of nonmycorrhizal controls at low fertility. Plant development was also stimulated by G. margarita at the high fertility level (3.54 gm 10-10-10 N-P-K/pot), but the magnitude of the increase was not as great as that at the low fertility level. Although cotton was a suitable host for Pratylenchus brachyurus, plant development was not retarded by this nematode at either fertility level. In concomitant culture, mycorrhizal-induced plant growth, and sporulation of the endomycorrhizal fungus were not affected by P. brachyurus. Reproduction of P. brachyurus was similar on mycorrhizal and nonmycorrhizal cotton.     

Spilanthes acmella ethanolic flower extract: LC-MS alkylamide profiling and its effects on sexual behavior in male rats

According to Indian Systems of Medicine, Spilanthes acmella (L.) Murr. (Family - Asteraceae), is considered effective in the treatment of sexual deficiencies especially due to ageing. In the present study, characterization of ethanolic extracts of the Spilanthes acmella flower and its effect on general mating pattern, penile erection and serum hormone levels of normal male Wistar albino rats were investigated and compared with sildenafil citrate. In vitro nitric oxide release was also investigated in human corpus cavernosum cell line. As N-alkylamides are a promising group, their profiling was performed using a gradient reversed phase high performance liquid chromatography/electrospray ionization ion trap mass spectrometry (HPLC/ESI-MS) method on an embedded polar column. MS¹ and MS² fragmentation data were used for identification purposes. For assessment of sexual behavior, animals were divided into five groups of eight male rats. The extracts (50, 100 and 150 mg/kg body weight/day) and sildenafil citrate (5 mg/kg body weight/day) (positive control) were administered orally for 28 days. The behavioral and sexual parameters were observed at days 0, 15, 28 and after a lapse of 7 and 14 days of discontinuance of drug treatment. Five N-isobutylamides, one 2-methylbutylamide and one 2-phenylethylamide were identified. The orally administered extract had a dose dependent positive effect on mounting frequency, intromission frequency and ejaculation frequency and the most significant effects (p < 0.05) were observed at 150 mg/kg treatment, even after a lapse of 7 and 14 days of discontinuance of drug treatment. A dose dependent effect was also observed on the FSH, LH and testosterone serum levels. With 150 mg/kg of ethanolic extract the values for FSH, LH and testosterone were 3.10 ± 0.25 mlU/ml, 6.87 ± 0.18 mlU/ml and 3.72 ± 0.12 ng/ml, respectively. In vitro nitric oxide release was 21.7 ± 2.9 μM, which was significantly higher compared to the control group (p < 0.01). Sildenafil citrate exhibited also a significant effect on NO release, but no effect on hormone levels of rats was observed. The aphrodisiac potential of an ethanolic Spilanthes acmella extract was demonstrated in vitro and in vivo. N-Alkylamides might attribute to the improved sexual potential. Study lends support to the traditional utilization of S. acmella as a sexual stimulating agent.     

Cryopreservation of the Mediterranean mussel (Mytilus galloprovincialis) spermatozoa

The effects of cryoprotectants, cooling rate and freezing on the mussel Mytilus galloprovincialis sperm were evaluated. At the end of each step of the experimental protocol, motility and fertilization ability of sperm were analyzed, compared to fresh semen. Five cryoprotectants were tested in their toxicity level: dimethylsulfoxide, ethylene glycol, 1-2 propylene glycol at 5%, 7%, 10%, 15% and 20% concentration; glycerol and methanol at concentration of 5%, 7% and 10%. The incubation times were 10, 20 and 30 min at 20 ± 1 °C. Only dimethylsulfoxide, ethylene glycol and 1-2 propylene glycol at 5%, 7% and 10% were chosen for the following pre-freezing step. Five adaptation/chilling rates were analyzed: 10 min at 20 ± 1, −2, −1, −0.5 and −0.25 °C/min and the last one was used for testing the best freezing procedure among seven gradients. Particularly, two rapid rates, three slow rates and two double step rates were conducted.Thawing results showed that M. galloprovincialis sperm are very sensitive to rapid pre-freezing and freezing protocols and only a slow procedure assured good motility and fertilization percentages.