Rapid quantification of plant-powdery mildew interactions by qPCR and conidiospore counts

ABSTRACT: BACKGROUND: The powdery mildew disease represents a valuable patho-system to study the interaction between plant hosts and obligate biotrophic fungal pathogens. Numerous discoveries have been made on the basis of the quantitative evaluation of plant-powdery mildew interactions, especially in the context of hyper-susceptible and/or resistant plant mutants. However, the presently available methods to score the pathogenic success of powdery mildew fungi are laborious and thus not well suited for medium- to high-throughput analysis. RESULTS: Here we present two new protocols that allow the rapid quantitative assessment of powdery mildew disease development. One procedure depends on quantitative polymerase chain reaction (qPCR)-based evaluation of fungal biomass, while the other relies on the quantification of fungal conidiospores. We validated both techniques using the powdery mildew pathogen Golovinomyces orontii on a set of hyper-susceptible and resistant Arabidopsis thaliana mutants and found that both cover a wide dynamic range of one to two (qPCR) and four to five (quantification of conidia) orders of magnitude, respectively. The two approaches yield reproducible results and are easy to perform without specialized equipment. CONCLUSIONS: The qPCR and spore count assays rapidly and reproducibly quantify powdery mildew pathogenesis. Our methods are performed at later stages of infection and discern mutant phenotypes accurately. The assays therefore complement currently used procedures of powdery mildew quantification and can overcome some of their limitations. In addition, they can easily be adapted to other plant-powdery mildew patho-systems.     

Cutaneous epitheliotropic T‐cell lymphoma with dissemination to the liver in an eastern chipmunk (Tamias striatus)

Cutaneous epitheliotropic T‐cell lymphoma with liver metastasis was diagnosed in a 10‐year‐old eastern chipmunk (Tamias striatus). Physical findings included intracutaneous swellings, ulcerated plaques and nodules, hypotrichosis and erythema of the skin. Fine needle aspiration from the skin lesions showed a population of large lymphocytes and lymphoblasts, and was helpful in establishing the diagnosis antemortem. The post‐mortem examination revealed epitheliotropic lymphoma with liver metastasis. Immunohistochemistry proved the T‐cell origin both in the liver and skin tumours. Electron microscopy did not reveal any viral particles within the tumour. To the authors’ knowledge, this is the first case of lymphoma described in this species.     

Toxicity of spiromesifen to the developmental stages of Bemisia tabaci biotype B

BACKGROUND: Spiromesifen is a novel insecticidal/acaricidal compound derived from spirocyclic tetronic acids that acts effectively against whiteflies and mites via inhibition of acetyl‐CoA‐carboxylase, a lipid metabolism enzyme. The effects of spiromesifen on the developmental stages of the whitefly Bemisia tabaci (Gennadius) were studied under laboratory conditions to generate baseline action thresholds for field evaluations of the compound. RESULTS: Adult B. tabaci mortality rate after spiromesifen treatment (5 mg L^?1) was 40%. Treatment with 0.5 mg L^?1 reduced fecundity per female by more than 80%, and fertility was almost nil. LC₅₀ for eggs was 2.6 mg L^?1, and for first instar 0.5 mg L^?1. Scanning electron microscopy revealed that eggs laid by treated adult females had an abnormally perforated chorion, and females were unable to complete oviposition. Light and fluorescent microscopy showed significantly smaller eggs following treatment, and smaller, abnormally formed and improperly localized bacteriomes in eggs and nymphs. The number of ovarioles counted in females treated with 5 mg L^?1 was significantly reduced. Spiromesifen showed no cross‐resistance with other commonly used insecticides from different chemical groups, and resistance monitoring in Israel showed no development of field resistance to this insecticide after 1 year of use. CONCLUSION: The strong effect on juvenile stages of B. tabaci with a unique mode of action and the absence of cross‐resistance with major commonly used insecticides from different chemical groups suggest the use of spiromesifen in pest and resistance management programmes. Copyright © 2008 Society of Chemical Industry     

Neonicotinoid resistance in rice brown planthopper, Nilaparvata lugens

BACKGROUND: Rice brown planthopper, Nilaparvata lugens Stål, is a primary insect pest of cultivated rice, and effective control is essential for economical crop production. Resistance to neonicotinoid insecticides, in particular imidacloprid, has been reported as an increasing constraint in recent years. In order to investigate the extent of resistance, 24 samples of N. lugens were collected from China, India, Indonesia, Malaysia, Thailand and Vietnam during 2005 and 2006. Their responses to two diagnostic doses of imidacloprid (corresponding approximately to the LC₉₅ and 5 × LC₉₅ of a susceptible strain) were examined. RESULTS: Ten of the 12 samples collected during 2005 were found to be susceptible to imidacloprid, but two late‐season samples from India showed reduced mortality at both diagnostic doses. All 13 strains collected in 2006 showed reduced mortality at both doses when compared with the susceptible strain. Dose–response lines showed resistance in one of the most resistant field strains to be approximately 100‐fold compared with the susceptible standard. CONCLUSION: The data demonstrate the development and spread of neonicotinoid resistance in N. lugens in Asia and support reports of reduced field efficacy of imidacloprid. Copyright © 2008 Society of Chemical Industry     

Cloning of <Emphasis Type="Bold">β</Emphasis>-tubulin and succinate dehydrogenase genes from <Emphasis Type="Italic">Uromyces fabae</Emphasis> and establishing selection conditions for their use in transformation

The obligate biotrophic nature of rust fungi calls for an in planta selection scheme as a means of developing a rust transformation technology. We show that the fungicides benomyl (used as its formulated product benlate) and carboxin suppress morphogenesis of the rust fungus Uromyces fabae in vitro and disease in planta, the latter without affecting the health of the host. The limits of their applicability were determined regarding concentration, method of application and optimal time intervals of treatment. Besides procedures for selection, a stable transformation system will also need to include genetic markers allowing to enrich for transformed cells within a large background of untransformed cells. Since the molecular targets of benlate and carboxin had been identified as -tubulin and succinate dehydrogenase, respectively, the corresponding genes (Uf-TBBIand Uf-SUCDHI) were cloned and characterized. Molecular phylogenies demonstrate that both are typical homologs to those of other Basidiomycota. RT-PCR analysis confirmed that both genes are constitutively expressed in all developmental stages of the mitotic uredospore multiplication cycle. Since homologs of Uf-TBB1and Uf-SUCDH1 have been successfully used as selection markers in other fungal systems, they provide valuable tools to develop additional corner stones of a stable transformation system for rust fungi.     

On the mode of action of the herbicides cinmethylin and 5-benzyloxymethyl-1, 2-isoxazolines: putative inhibitors of plant tyrosine aminotransferase

BACKGROUND: The mode of action of the grass herbicides cinmethylin and 5‐benzyloxymethyl‐1,2‐isoxazolines substituted with methylthiophene (methiozolin) or pyridine (ISO1, ISO2) was investigated. RESULTS: Physiological profiling using a series of biotests and metabolic profiling in treated duckweed (Lemna paucicostata L.) suggested a common mode of action for the herbicides. Symptoms of growth inhibition and photobleaching of new fronds in Lemna were accompanied with metabolite changes indicating an upregulation of shikimate and tyrosine metabolism, paralleled by decreased plastoquinone and carotenoid synthesis. Supplying Lemna with 10 µM of 4‐hydroxyphenylpyruvate (4‐HPP) reversed phytotoxic effects of cinmethylin and isoxazolines to a great extent, whereas the addition of L ‐tyrosine was ineffective. It was hypothesised that the herbicides block the conversion of tyrosine to 4‐HPP, catalysed by tyrosine aminotransferase (TAT), in the prenylquinone pathway which provides plastoquinone, a cofactor of phytoene desaturase in carotenoid synthesis. Accordingly, enhanced resistance to ISO1 treatment was observed in Arabidopsis thaliana L. mutants, which overexpress the yeast prephenate dehydrogenase in plastids as a TAT bypass. In addition, the herbicides were able to inhibit TAT7 activity in vitro for the recombinant enzyme of A. thaliana. CONCLUSION: The results suggest that TAT7 or another TAT isoenzyme is the putative target of the herbicides. Copyright © 2011 Society of Chemical Industry     

Composition of saturnian stream particles

During Cassini's approach to Saturn, the Cosmic Dust Analyser (CDA) discovered streams of tiny (less than 20 nanometers) high-velocity (approximately 100 kilometers per second) dust particles escaping from the saturnian system. A fraction of these impactors originated from the outskirts of Saturn's dense A ring. The CDA time-of-flight mass spectrometer recorded 584 mass spectra from the stream particles. The particles consist predominantly of oxygen, silicon, and iron, with some evidence of water ice, ammonium, and perhaps carbon. The stream particles primarily consist of silicate materials, and this implies that the particles are impurities from the icy ring material rather than the ice particles themselves.     

Incidence and characterisation of resistance to neonicotinoid insecticides and pymetrozine in the greenhouse whitefly,Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae)

BACKGROUND: Trialeurodes vaporariorum (Westwood), also known as the greenhouse whitefly, is a serious pest of protected vegetable and ornamental crops in most temperate regions of the world. Neonicotinoid insecticides are used widely to control this species, although resistance has been reported and may be becoming widespread. RESULTS: Mortality rates of UK and European strains of T. vaporariorum to a range of neonicotinoids and pymetrozine, a compound with a different mode of action, were calculated, and significant resistance was found in some of those strains. A strong association was found between neonicotinoids and pymetrozine, and reciprocal selection experiments confirmed this finding. Expression of resistance to the neonicotinoid imidacloprid and pymetrozine was age specific, and resistance in nymphs did not compromise recommended application rates. CONCLUSION: This study indicates strong parallels in the phenotypic characteristics of neonicotinoid resistance in T. vaporariorum and the tobacco whitefly Bemisia tabaci Gennadius, suggesting possible parallels in the underlying mechanisms. Copyright © 2010 Society of Chemical Industry     

Modelling approaches to compare sorption and degradation of metsulfuron-methyl in laboratory micro-lysimeter and batch experiments

Results of laboratory batch studies often differ from those of outdoor lysimeter or field plot experiments--with respect to degradation as well as sorption. Laboratory micro-lysimeters are a useful device for closing the gap between laboratory and field by both including relevant transport processes in undisturbed soil columns and allowing controlled boundary conditions. In this study, sorption and degradation of the herbicide metsulfuron-methyl in a loamy silt soil were investigated by applying inverse modelling techniques to data sets from different experimental approaches under laboratory conditions at a temperature of 10 degrees C: first, batch-degradation studies and, second, column experiments with undisturbed soil cores (28 cm length x 21 cm diameter). The column experiments included leachate and soil profile analysis at two different run times. A sequential extraction method was applied in both study parts in order to determine different binding states of the test item within the soil. Data were modelled using ModelMaker and Hydrus-1D/2D. Metsulfuron-methyl half-life in the batch-experiments (t1/2 = 66 days) was shown to be about four times higher than in the micro-lysimeter studies (t1/2 about 17 days). Kinetic sorption was found to be a significant process both in batch and column experiments. Applying the one-rate-two-site kinetic sorption model to the sequential extraction data, it was possible to associate the stronger bonded fraction of metsulfuron-methyl with its kinetically sorbed fraction in the model. Although the columns exhibited strong significance of multi-domain flow (soil heterogeneity), the comparison between bromide and metsulfuron-methyl leaching and profile data showed clear evidence for kinetic sorption effects. The use of soil profile data had significant impact on parameter estimates concerning sorption and degradation. The simulated leaching of metsulfuron-methyl as it resulted from parameter estimation was shown to decrease when soil profile data were considered in the parameter estimation procedure. Moreover, it was shown that the significance of kinetic sorption can only be demonstrated by the additional use of soil profile data in parameter estimation. Thus, the exclusive use of efflux data from leaching experiments at any scale can lead to fundamental misunderstandings of the underlying processes.