Obesity wars: molecular progress confronts an expanding epidemic

The worldwide prevalence of obesity is increasing at an alarming rate, with major adverse consequences for human health. This "obesity epidemic" is paralleled by a rapid and substantive increase in our understanding of molecular pathways and physiologic systems underlying the regulation of energy balance. While efforts to address the environmental factors that are responsible for the recent "epidemic" must continue, new molecular and physiologic insights into this system offer exciting possibilities for future development of successful therapies.     

Biological control of Botrytis gray mould and Sclerotinia drop in lettuce

Research was carried out to evaluate the effectiveness of the biological control of two most important fungal diseases of lettuce (Lactuca sativa L.): 1) Botrytis Gray Mould caused by Botrytis cinerea Pers. ex Fr.; 2) Sclerotinia Drop caused by two pathogenic fungi, Sclerotinia sclerotiorum (Lib.) De Bary and/or Sclerotinia minorJagger. Biological control in lettuce was carried out: 1) using Coniothyrium minitans Campbell, an antagonist fungus that attacks and destroys sclerotia within the soil; 2) identifying lettuce genotypes showing less susceptibility or tolerance. The object of this research was to find control strategies to reduce chemical treatments. The use of resistant varieties is one of the most economical ways to control vegeTable diseases. The lettuce genotypes showing in preliminary trials the best behaviour to the sclerotial diseases were compared in a randomized complete block experiment design and replicated four times. Observations were carried out from February up to April registering the number of diseased plants and yield. The pathogens were isolated on PDA medium and identified. The isolates grown onto PDA plates, after incubation for 6 weeks, allowed obtaining sclerotia that were the target of C. minitans in biological control trials. In laboratory, in controlled conditions, 27 small plots (30 cm in diameter each) with disinfected soil were performed. In 18 plots 9 sclerotia were inoculated (per plot, three of each fungus) and in 9 plots of them a suspension of the antagonist fungus was added. Subsequently, three lettuce varieties were transplanted. For each variety were compared: 1) untreated plots; 2) treated plots with sclerotia only; 3) treated plots with sclerotia and C. minitans suspension. The number of diseased plants was recorded. According to symptom evaluation scale, ranged from 0 (no disease) up to 10 (100% necrotic leaves or dead plants) the plants were grouped into infection classes, calculating the McKinney index. In greenhouse trials, "LM 1307" genotype showed less significant susceptibility to Botrytis Gray Mould (0-2% of affected plants), while "Ninja" and "Charmy" showed 4-11% and 16-26% of diseased plants, respectively. The yields were 69.7, 62.7, 55.3 t/Ha, respectively. In laboratory tests, the McKinney index gave the following results: no disease in all untreated plants; 38.3, 54.2 and 89.2% in "LM 1307", "Ninja" and "Charmy" treated with sclerotia only, respectively; 2.5, 7.5 and 20.8% in "LM 1307", "Ninja" and "Charmy" treated with sclerotia and C. minitans, respectively. In conclusion, the less susceptibility of the genotypes to sclerotial diseases and the use of hyperparasites of sclerotia of phytopathogenic fungi exhibited best results.     

Effect of turfgrass mowing height on biocontrol of dandelion with Sclerotinia minor

The fungus, Sclerotinia minor Jagger is under development as a bioherbicide for control of dandelion and many broadleaf weeds in turfgrass environments. The effect of S. minor on dandelion survival was evaluated under different mowing heights and compared with a commonly used herbicide Killex™. In the greenhouse, the onset of symptoms was more rapid, foliar damage was more severe, and the reduction of aboveground biomass and root biomass was greater for the bioherbicide than the herbicide. The bioherbicide reduced root biomass ≥10-fold compared with untreated plants. Under high weed infestation levels in the field, S. minor caused a greater initial reduction of dandelion density than did the herbicide during the 2-week post-application period, although reductions were greater in herbicide treated plots by 6 weeks after application. Over the growing season, S. minor and the herbicide had similar suppressive effects on dandelion density except under the closest mowing height (3-5 cm). After treatment, close mowing favored dandelion seedling recruitment and the biocontrol had no residual activity. Survival of dandelion roots was significantly less after spring than fall treatment of S. minor and season long mowing at the close height significantly reduce root survival. Close mowing may be detrimental for S. minor applications on heavily infested domestic lawns and amenity grassland areas.     

Differentiation of Sclerotinia minor depends on thiol redox state and oxidative stress

Sclerotial differentiation in Sclerotinia minor is associated with oxidative stress and thiol redox state. The significance of oxidative stress to sclerotial differentiation was revealed by the higher oxidative stress of S. minor compared with a nonsclerotiogenic counterpart. The effect of thiol redox state on sclerotial differentiation was shown by the antioxidant action of the thiol (-SH) group of N-acetylcysteine and cysteine and by an unknown (not antioxidant) role of glutathione (GSH) on S. minor. The nonantioxidant role of GSH was indicated by the differentiation-inhibiting and differentiation-noninhibiting actions of the GSH biosynthesis inhibitor L-buthionine-S,R-sulfoximine and the GSH biosynthesis inducer L-2-oxo-thiazolidine-4-carboxylate, respectively, and by the increase of oxidative stress they caused during the transition from the undifferentiated to differentiated state of S. minor. Moreover, N-acetylcysteine can be used as a potent nontoxic fungicide against this phytopathogenic fungus by acting as a growth-inhibiting cytotoxic oxidant and by sustaining the fungus in the undifferentiated hyphal stage, which is vulnerable to degradation by soil microorganisms.     

Physiological characterization of the dandelion bioherbicide,Sclerotinia minor IMI 344141

The fungus Sclerotinia minor (IMI 344141) is being developed as a biological control for dandelion and other broadleaf weeds in turfgrass environments. Being a microbial pest control agent (MPCA), the S. minor strain must be characterized to show relatedness to like organisms and to distinguish the MPCA from related microorganisms. Phenotypic variation among 30 isolates of S. minor, collected from different regions and hosts, was studied on potato dextrose agar (PDA) and oatmeal agar (OMA). Isolates varied significantly in sclerotia shape (length/width ratio) and number, but did not vary in colony morphology or growth rates. There was high diversity (0.6) among the mycelial compatibility groups (MCG) as seven multi-member and 11 single member groups were recognized. Isolates were categorized into highly virulent, virulent, moderately virulent, and hypo virulent based on 48 h post mycelial growth on detached dandelion leaves. When assessed on dandelion plants in the greenhouse, isolate IMI 344141 ranked the highest in biocontrol efficacy, reduction of above- and below-ground biomass, and reduction in dandelion survival. Oxalic acid production was not correlated with isolate aggressiveness or growth rate and did not vary among isolates of the same MCG. IMI 344141 can be phenotypically distinguished from the other tested S. minor isolates by performing vegetative compatibility testing and counting sclerotia produced on standard 9-cm diameter PDA plates. IMI 344141 produces <100 sclerotia/plate.     

Enhanced degradation of iprodione in soil after repeated treatments for controlling Sclerotinia minor

Poor field control of lettuce collar rot by iprodione was observed in southern France and was attributed to enhanced biodegradation of the fungicide. Enhanced biodegradation was obtained in vitro after repeated applications of iprodione to non-degrading soils. Normal soils became biodegrading after mixing with degrading soils (3 vol./1 vol.). Activity of the responsible microflora seemed dependent on soil physico-chemical characteristics.     

Sclerotinia minor avances fruiting and reduces germination in dandelion (Taraxacum officinale)

Sclerotinia minor Jagger is a promising biocontrol agent for dandelion in turfgrass. When a flowering dandelion population was treated with S. minor, flowering accelerated to the fruiting stage within 4 days. This developmental response was 4-5 days earlier than in the control, untreated plants and was not observed in herbicide-treated plants. Seeds obtained from the fungal-treated plants were smaller, lighter and their germination rate was reduced by 48.4 and 47.3% for spring and fall applications, respectively. S. minor was not detected in dandelion seeds from the fungal-treated plants. In addition to effective control of mature (flowering) dandelions, seeds dispersed by dying plants have reduced germination and are not transferring S. minor off target.     

The role of ascorbic acid role in the differentiation of sclerotia in Sclerotinia minor

Sclerotinia minor in culture produces ascorbic acid in levels dependent on oxidative growth conditions and stage of development. During differentiation reduced/oxidized ascorbate ratio decreased by 12 and 6 fold at high and low oxidative stress, respectively. Exogenous ascorbate caused a concentration-dependent decrease of oxidative stress (lipid peroxidation), inhibition of sclerotial differentiation (up to 100%) and delay of differentiatlon (up to 10 days). Ascorbic acid may be produced to help the fungus reduce oxidative stress during growth. The data of this study support our theory proposing that oxidative stress is the inducing factor of sclerotial differentiation in fungi.This revised version was published online in October 2005 with corrections to the Cover Date.     

Development of strain specific molecular markers for the Sclerotinia minor bioherbicide strain IMI 344141

The plant pathogenic fungus, Sclerotinia minor IMI 344141, has been developed as a bioherbicide for broadleaf weed control in turfgrass and a means to differentiate this biocontrol agent from like organisms is required. A strain specific molecular marker was developed to detect and monitor the Sclerotinia minor IMI 344141 bioherbicide strain. The method was based on polymerase chain reaction (PCR) amplification of two sequence-characterized amplified regions (SCAR) primer pairs for a first round PCR, and another two sets of nested primers was used for a second round PCR if higher sensitivity was needed. Sclerotinia minor IMI 344141 was successfully traced from both pure cultures and environmental samples originating from bioherbicide-released field trials. DNA of the S. minor bioherbicide isolate IMI 344141 was detected in the soil 2 months after application, but was not detected in the 3- and 9-month samples after application. When applied as a bioherbicide, S. minor (IMI 344141) did not persist into the following spring season in turf environments. This molecular detection method provides a mechanism to distinguish this isolate from related organisms and a tool to monitor behavior of the biocontrol agent S. minor IMI 344141 in nature, particularly in soil.     

Enhancing resistance to Sclerotinia minor in peanut by expressing a barley oxalate oxidase gene

Sclerotinia minor Jagger is the causal agent of Sclerotinia blight, a highly destructive disease of peanut (Arachis hypogaea). Based on evidence that oxalic acid is involved in the pathogenicity of many Sclerotinia species, our objectives were to recover transgenic peanut plants expressing an oxalic acid-degrading oxalate oxidase and to evaluate them for increased resistance to S. minor. Transformed plants were regenerated from embryogenic cultures of three Virginia peanut cultivars (Wilson, Perry, and NC-7). A colorimetric enzyme assay was used to screen for oxalate oxidase activity in leaf tissue. Candidate plants with a range of expression levels were chosen for further analysis. Integration of the transgene was confirmed by Southern-blot analysis, and gene expression was demonstrated in transformants by northern-blot analysis. A sensitive fluorescent enzyme assay was used to quantify expression levels for comparison to the colorimetric protocol. A detached leaflet assay tested whether transgene expression could limit lesion size resulting from direct application of oxalic acid. Lesion size was significantly reduced in transgenic plants compared to nontransformed controls (65%-89% reduction at high oxalic acid concentrations). A second bioassay examined lesion size after inoculation of leaflets with S. minor mycelia. Lesion size was reduced by 75% to 97% in transformed plants, providing evidence that oxalate oxidase can confer enhanced resistance to Sclerotinia blight in peanut.     

Compost amendments enhance peat suppressiveness to Pythium ultimum,Rhizoctonia solani and Sclerotinia minor

Peat is the most common organic material used for the preparation of potting mix because of its homogeneous and favorable agronomic characteristics. However, this organic material is poorly suppressive against soilborne pathogens and fungicides are routinely used to manage damping-off diseases. In the present study, we investigated the suppressive capability of five compost – peat mixtures towards the plant pathogens Pythium ultimum, Rhizoctonia solani and Sclerotinia minor – Lepidium sativum pathosystems. For all organic media, 18 parameters were measured including enzymatic activities (glucanase, N-acetyl-glucosaminidase, chitobiosidase and hydrolysis of fluorescein diacetate), microbiological (BIOLOG® EcoPlates?, culturable bacteria and fungi), and chemical features (pH, EC, total, extractable and humic carbon, total and organic N, NH₄–N, total protein and water content). In addition, ¹³C-CPMAS-NMR spectroscopy was used to characterize the organic materials. Peat amended with composts reduced disease damping-off caused by P. ultimum, R. solani and S. minor in 60% of the mixtures and compost derived from animal manure showed the largest and most consistent disease suppression. Sterilization decreased or eliminated suppressiveness of 42.8% of the mixtures. The most useful parameters to predict disease suppression were different for each pathogen: extractable carbon, O-aryl C and C/N ratio for P. ultimum, alkyl/O-alkyl ratio, N-acetyl-glucosaminidase and chitobiosidase enzymatic activities for R. solani and EC for S. minor. Our results demonstrate that the addition of composts to peat could be useful for the control of soilborne pathogens.     

Resistance of Sclerotinia minor Isolates to Cyclic Imides in Lettuce Field Soils of Roussillon, France

Lettuce drop caused by Sclerotinia minor Jagger is the most common disease affecting lettuce crops during the fall and winter in Roussillon. Spraying of cyclic imide fungicides (iprodione, vinclozolin, and less commonly procymidone) generally provides satisfactory protection. However, since about 1985, the mean field efficacy of iprodione and vinclozolin has significantly decreased, whereas that of procymidone is currently not in question. The appearance of fungicide-resistant isolates of S.minor could explain the reduced efficacy of these fungicides. We thus looked for iprodione and procymidone-resistant isolates of S.minor in these problem fields. The investigations were earned on for over 3 years. Most of the isolates showed fungicide sensitivity (iprodione IC 50 = 0.17 mg 1⁻¹), about one quarter showed average resistance (iprodione IC 50 = 0.59 mg 1⁻¹) and a few were found to be highly resistant. Resistant isolates were also obtained in vitro when using high fungicide concentrations. Moreover, isolates resistant to iprodione were found to be resistant to procymidone. The most highly resistant isolates produced fewer and larger-sized sclerotia than the sensitive isolates, they were also more quickly inhibited by high osmotic pressures. Field variability was quite low: 73 of 74 isolates obtained from one field were found to be sensitive, with only one showing mild resistance. There did not seem to be any correlation between the observed decrease in the field efficacy of iprodione and the presence of resistant isolates.     

Inhibition of growth of Sclerotinia minor and other pathogens by citrinin in the filtrate of Penicillium citrinum

Penicillium citrinum Thom, isolated from the sclerotia of Sderotinia minor, was cultured in a broth of Czapek-Dox for 4 to 8 weeks. The filtrate obtained was incorporated into potato dextrose agar or Czapek-Dox agar at different concentrations (v/v). The amended media were tested for mycelial growth of S. minor and other pathogens. Mycelial growth of S. minor was completely inhibited on media amended with 20% (v/v) filtrate of P. citrinum, and considerable inhibition of S. minor occurred at 10 and 15% concentrations. Mycelial growth of S. major, Sclerotium rolfsii, Rhizoctonia solani (AG-4) was inhibited by similar concentrations of filtrate of P. citrinum. Inhibitor(s) in the filtrate were extracted with ethyl acetate and tentatively identified as citrinin. Citrinin was shown to be an active component in the filtrate against mycelial growth of S. minor, S. major and Sclerotium rolfsii.Cooperative investigation of U.S. Department of Agriculture, Agriculture Research Service and Oklahoma State University. Journal Article No. 4989, Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by USDA or by Oklahoma State University, or imply their approval to the exclusion of other products or vendors that may also be suitable.