Combining flow cytometry and gfp reporter gene for quantitative evaluation of Pectpbacterium carotovorum ssp. carotovorum in Ornithogalum dubium plantlets.

AIMS: Ornithogalum dubium is a natural host of the soft rot pathogen Pectobacterium carotovorum ssp. carotovorum (Pcc). The present study was aimed to develop a quantification system for Pcc expressing a gfp reporter gene, using fluorescent activated cell sorter (FACS) in planta. METHODS AND RESULTS: Several calibration steps were required to distinctly gate the GFP-labelled bacteria at FL1 mode and count the bacteria. To validate the bacterial counts obtained by FACS analysis, an internal standard of polystyrene green fluorescent microsphere beads was employed, resulting in high correlation with serial dilutions and plate counting. This allowed quantification of the bacteria, with no further need to culture, dilute or plate the cells. Micropropagation tools were developed to produce uniform plantlets of O. dubium, which were either inoculated with increasing concentrations of Pcc or elicited for resistance towards Pcc using methyl jasmonate. The rapid counting procedure allowed recovering, gating and counting the bacterial population in planta, separately from the plant cells background and from the microsphere beads. CONCLUSIONS: The FACS based quantification approach of Pcc was found accurate, reproducible and time saving, thus useful for counting bacteria in planta. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of time- and cost-saving approach for Pcc quantification with efficient screening tools during early stages of micropropagation may facilitate the preliminary process of selection for resistant cultivars.

The use of medicinal herbs by diabetic Jordanian patients.

Diabetes mellitus is the most common metabolic disorder worldwide. To date, there have been no reports on the frequency of use of herb medicines in the managements of diabetes mellitus in Jordan. This cross-sectional study was conducted by interviewing 310 diabetic patients visiting two medical centers in Jordan: Jordan University of Science & Technology Medical Center and Sarih Medical Center between December 2003 and August 2004. It is found that 31% of interviewed patients have used herbal products (96 patients). The results revealed that the most commonly used herbs by diabetic patients in Jordan were Trigonella foenumgraecum (22.9%), Lupinus albus (14.6%), Allium sativum (11.5%), Allium cepa (5.2%), Nigella sativa (7.3%), Zea mays L. (6.3%), Urtica dioica L. (8.3%), Eucalyptus globules LA (9.4%), Olea europea L. (3.1%), Cumminum cyminum (9.4%), Coriandrum sativum (10.4%), Salvia officinalis L. (3.1%), and Tilia cordata (1%). Furthermore, it is found that 47.9% of the patients used herbs according to advice from their friends on a daily basis. The side effects were reported by 36.5% of the patients and include headache, nausea, dizziness, itching, palpitation, and sweating. Among the patients, 72.9% used the herbs as adjunctive therapy along with their anti-diabetic drugs and 80.2% of the patients informed their physicians about their use. A 79.2% of the sample confirmed their intention to re-use these herbs as 86.5% of them were satisfied with their diabetes control. There was a significant relationship between the use of herbs, the patient's place of residence and his/her level of education. The main conclusion of this survey is that the use of medicinal herbs among diabetic patient in Jordan is common. Therefore, it is essential to increase the level of awareness among diabetic patients and health care providers regarding the efficacy and toxicity of these medicinal herbs.

Analyses of avocado (Persea americana) nectar properties and their perception by honey bees (Apis mellifera).

Honey bees are important avocado pollinators. However, due to the low attractiveness of flowers, pollination is often inadequate. Previous work has revealed that avocado honey is relatively unattractive to honey bees when compared with honey from competing flowers. We characterized avocado honey and nectar with respect to their odor, color, and composition of sugars, phenolic compounds, and minerals. Furthermore, we tested how honey bees perceive these parameters, using the proboscis extension response bioassay and preference experiments with free-flying bees. Naïve bees were indifferent to odors of avocado and citrus flowers and honey. Experienced bees, which were collected in the field during the blooming season, responded preferentially to odor of citrus flowers. The unique sugar composition of avocado nectar, which contains almost exclusively sucrose and a low concentration of the rare carbohydrate perseitol, and the dark brown color of avocado honey, had no negative effects on its attractiveness to the bees. Phenolic compounds extracted from avocado honey were attractive to bees and adding them to a solution of sucrose increased its attractiveness. Compared with citrus nectar and nonavocado honey, avocado nectar and honey were rich in a wide range of minerals, including potassium, phosphorus, magnesium, sulfur, iron, and copper. Potassium and phosphorus, the two major minerals, both had a repellent effect on the bees. Possible explanations for the presence of repellent components in avocado nectar are discussed.

Tomato fruit size, maturity and alpha-tomatine content influence the performance of larvae of potato tuber moth Phthorimaea operculella (Lepidoptera: Gelechiidae).

Various physical and chemical properties of host plants influence insect larval performance and subsequent adult fitness. Tomato plants are relatively new hosts to the potato tuber moth, Phthorimaea operculella (Zeller), with the fruit being its preferred feeding site. However, it is unclear how the biochemical and physical properties of tomato fruits relate to potato tuber moth performance. Significant amounts of alpha-tomatine were detected in maturing green and ripening fruits of cherry (cv. Ceres) and processing (cv. Serio) types of tomatoes whereas none was detected in a fresh market variety (cv. Marglobe), at comparable stages. alpha-Tomatine is negatively and significantly correlated with development rate (head capsule size) of larvae reared in the fruits of the cherry and processing type tomatoes. Generally, survival, growth and development were significantly superior for larvae reared in the ripening fruits of the fresh market cultivar. At this stage, the fruits of this cultivar are also the largest. Based on these results it is concluded that fruit alpha-tomatine content, as well as fruit size and maturity, all affect performance of P. operculella larvae in the fruits of cultivated tomatoes.

Biodegradative mechanism of the brown rot basidiomycete Gloeophyllum trabeum: evidence for an extracellular hydroquinone-driven fenton reaction.

We have identified key components of the extracellular oxidative system that the brown rot fungus Gloeophyllum trabeum uses to degrade a recalcitrant polymer, polyethylene glycol, via hydrogen abstraction reactions. G. trabeum produced an extracellular metabolite, 2,5-dimethoxy-1,4-benzoquinone, and reduced it to 2,5-dimethoxyhydroquinone. In the presence of 2,5-dimethoxy-1,4-benzoquinone, the fungus also reduced extracellular Fe3+ to Fe2+ and produced extracellular H2O2. Fe3+ reduction and H2O2 formation both resulted from a direct, non-enzymatic reaction between 2,5-dimethoxyhydroquinone and Fe3+. Polyethylene glycol depolymerization by G. trabeum required both 2,5-dimethoxy-1,4-benzoquinone and Fe3+ and was completely inhibited by catalase. These results provide evidence that G. trabeum uses a hydroquinone-driven Fenton reaction to cleave polyethylene glycol. We propose that similar reactions account for the ability of G. trabeum to attack lignocellulose.

Rapid polyether cleavage via extracellular one-electron oxidation by a brown-rot basidiomycete.

Fungi that cause brown rot of wood are essential biomass recyclers and also the principal agents of decay in wooden structures, but the extracellular mechanisms by which they degrade lignocellulose remain unknown. To test the hypothesis that brown-rot fungi use extracellular free radical oxidants as biodegradative tools, Gloeophyllum trabeum was examined for its ability to depolymerize an environmentally recalcitrant polyether, poly(ethylene oxide) (PEO), that cannot penetrate cell membranes. Analyses of degraded PEOs by gel permeation chromatography showed that the fungus cleaved PEO rapidly by an endo route. 13C NMR analyses of unlabeled and perdeuterated PEOs recovered from G. trabeum cultures showed that a major route for depolymerization was oxidative C---C bond cleavage, a reaction diagnostic for hydrogen abstraction from a PEO methylene group by a radical oxidant. Fenton reagent (Fe(II)/H2O2) oxidized PEO by the same route in vitro and therefore might account for PEO biodegradation if it is produced by the fungus, but the data do not rule out involvement of less reactive radicals. The reactivity and extrahyphal location of this PEO-degrading system suggest that its natural function is to participate in the brown rot of wood and that it may enable brown-rot fungi to degrade recalcitrant organopollutants.

Effect of manganese on preferential degradation of lignin by Pleurotus ostreatus during solid-state fermentation.

Practical utilization of the polysaccharides in the lignocellulosic complex is limited because of the high lignin content of the complex. In this study we focused on the effect of Mn on lignin and cellulose biodegradation during solid-state fermentation by the edible mushroom Pleurotus ostreatus. Preferential degradation of lignin was enhanced by the addition of Mn(II) to cotton stalks at concentrations ranging from 30 to 620 micrograms of Mn per g. This effect was most apparent when we compared mineralization rates of [14C] lignin with mineralization rates of [14C] cellulose. Enhanced selectivity was also observed when we analyzed residual organic matter at the end of the fermentation period by using crude fiber analysis. The cellulose fraction in the original material was 1.8 times larger than the cellulose fraction of lignin. The cellulose/lignin ratio increased during 32 days of solid-state fermentation from 2.5 in the control to 3.3 following the addition of Mn to the medium. The in vitro digestibility value for fermented cotton stalks was 53% of the dry matter. Addition of 600 micrograms of Mn per g to the cotton stalks resulted in a digestibility value of 65.4%. Enhancement of preferential lignin degradation could be result of either increased activity of the ligninolytic enzymes or production of Mn (III), which might preferentially degrade aromatic structures in the lignocellulosic complex.

Effect of Manganese on Lignin Degradation by Pleurotus ostreatus during Solid-State Fermentation.

Lignin degradation by Pleurotus ostreatus was studied under solid-state fermentation (SSF) in chemically defined medium containing various levels of Mn. Degradation of [C]lignin prepared from cotton branches to soluble products, as well as its mineralization to CO(2), was enhanced by the addition of Mn. The effect of malonate on lignin mineralization was most marked during the first 10 days of SSF, in a treatment amended with 73 muM Mn. A high concentration of Mn (4.5 mM) caused inhibition of both fungal growth and mineralization rates during the first 2 weeks of incubation. Addition of malonate reversed this effect because of chelation of Mn. Mn was found to precipitate in all treatments, with or without the addition of malonate. alpha-Keto-gamma-methiolbutyric acid cleavage to ethylene, an indication of OH production, was observed as early as 3 days of incubation in all treatments.

Lignocellulose Degradation during Solid-State Fermentation: Pleurotus ostreatus versus Phanerochaete chrysosporium.

Lignocellulose degradation and activities related to lignin degradation were studied in the solid-state fermentation of cotton stalks by comparing two white rot fungi, Pleurotus ostreatus and Phanerochaete chrysosporium. P. chrysosporium grew vigorously, resulting in rapid, nonselective degradation of 55% of the organic components of the cotton stalks within 15 days. In contrast, P. ostreatus grew more slowly with obvious selectivity for lignin degradation and resulting in the degradation of only 20% of the organic matter after 30 days of incubation. The kinetics of C-lignin mineralization exhibited similar differences. In cultures of P. chrysosporium, mineralization ceased after 18 days, resulting in the release of 12% of the total radioactivity as CO(2). In P. ostreatus, on the other hand, 17% of the total radioactivity was released in a steady rate throughout a period of 60 days of incubation. Laccase activity was only detected in water extracts of the P. ostreatus fermentation. No lignin peroxidase activity was detected in either the water extract or liquid cultures of this fungus. 2-Keto-4-thiomethyl butyric acid cleavage to ethylene correlated to lignin degradation in both fungi. A study of fungal activity under solid-state conditions, in contrast to those done under defined liquid culture, may help to better understand the mechanisms involved in lignocellulose degradation.