Comparison of methods to detect resistance of Helminthosporium solani to a thiabendazole fungicide

Le développement de la résistance à un fongicide de benzimidazole, le thiabendazole (TBZ), utilisé après la récolte et avant la plantation, a eu comme conséquence l’augmentation de l’incidence de la tache argentée de la pomme de terre causée par Helminthosporium solani. Des analyses conventionnelles ou basées sur la réaction de polymérisation en chaîne (PCR) ont été comparées pour détecter, parmi 54 isolats de H. solani, les isolats sensibles (TBZS) et résistants (TBZR) au TBZ. L’analyse basée sur la PCR a déterminé les 27 isolats simples de spores de l’est du Canada, neuf des isolats de l’Alberta (ouest du Canada) et six des isolats du North Dakota (États-Unis) comme TBZR, le reste des isolats étant TBZS. Les résultats de l'analyse basée sur la PCR ont été bien corrélés avec l’évaluation de la sensibilité au TBZ des isolats de H. solani sur le support avec amendement au TBZ. En outre, la détection de TBZR- H. solani dans 20 échantillons de tubercules infectés par la tache argentée par l’analyse basée sur la PCR démontre que cette méthode peut être utilisée dans la détection de l’agent pathogène directement à partir des lésions infectées du tubercule. La méthode basée sur la PCR était plus rapide (1 jour) que la technique conventionnelle (5 semaines).The development of resistance to a post-harvest and preplanting benzimidazole fungicide, thiabendazole (TBZ), has resulted in the increase of silver scurf of potato caused by Helminthosporium solani. Conventional and polymerase chain reaction (PCR)-based assays were compared, among 54 isolates of H. solani, to detect the TBZ-sensitive (TBZS) and -resistant (TBZR) isolates. In a PCR-based assay, all 27 single-spore isolates from eastern Canada, nine isolates from Alberta in western Canada, six isolates from North Dakota, USA were distinguished as TBZR, the remainder of the isolates being determined as TBZS. The results from the PCR-based assay correlated well with the TBZ sensitivity assessment of isolates of H. solani on the TBZ-amended medium. Also, the detection of TBZR- H. solani in 20 tuber samples with silver scurf lesions by the PCR-based assay demonstrates that this method can be used to detect the pathogen directly from the tuber lesions. The PCR-based method was more rapid (1 day) than the conventional technique (5 weeks)

Embryonic Lethals and T-DNA Insertional Mutagenesis in Arabidopsis

T-DNA insertional mutagenesis represents a promising approach to the molecular isolation of genes with essential functions during plant embryo development. We describe in this report the isolation and characterization of 18 mutants of Arabidopsis thaliana defective in embryo development following seed transformation with Agrobacterium tumefaciens. Random T-DNA insertion was expected to result in a high frequency of recessive embryonic lethals because many target genes are required for embryogenesis. The cointegrate Ti plasmid used in these experiments contained the nopaline synthase and neomycin phosphotransferase gene markers. Nopaline assays and resistance to kanamycin were used to estimate the number of functional inserts present in segregating families. Nine families appeared to contain a T-DNA insert either within or adjacent to the mutant gene. Eight families were clearly not tagged with a functional insert and appeared instead to contain mutations induced during the transformation process. DNA gel blot hybridization with internal and right border probes revealed a variety of rearrangements associated with T-DNA insertion. A general strategy is presented to simplify the identification of tagged embryonic mutants and facilitate the molecular isolation of genes required for plant embryogenesis

GFP-tagged multimetal-tolerant bacteria and their detection in the rhizosphere of white mustard

The introduction of rhizobacteria that tolerate heavy metals is a promising approach to support plants involved in phytoextraction and phytostabilisation. In this study, soil of a metal-mine wasteland was analyzed for the presence of metal-tolerant bacterial isolates, and the tolerance patterns of the isolated strains for a number of heavy metals and antibiotics were compared. Several of the multimetal-tolerant strains were tagged with a broad host range reporter plasmid (i.e. pPROBE-NT) bearing a green fluorescent protein marker gene (gfp). Overall, the metal-tolerant isolates were predominately Gram-negative bacteria. Most of the strains showed a tolerance to five metals (Zn, Cu, Ni, Pb and Cd), but with differing tolerance patterns. From among the successfully tagged isolates, we used the transconjugant Pseudomonas putida G25 (pPROBE-NT) to inoculate white mustard seedlings. Despite a significant decrease in transconjugant abundance in the rhizosphere, the gfp-tagged cells survived on the root surfaces at a level previously reported for root colonisers

Cross-resistance between myclobutanil and tebuconazole and the genetic basis of tebuconazole resistance in Venturia inaequalis

BACKGROUND: Myclobutanil is one of the most widely used demethylation inhibitor (DMI) fungicides for the management of apple scab, caused by Venturia inaequalis. Strains of V. inaequalis resistant to myclobutanil have been reported across the world. Tebuconazole, another DMI fungicide, has been proposed as an alternative to myclobutanil, and the extent of cross-resistance with myclobutanil therefore needs to be evaluated. The sensitivity to tebuconazole and myclobutanil of a total of 40 isolates was determined. Half the isolates came from an isolated orchard which had never been sprayed with fungicides and half from orchards sprayed regularly with myclobutanil, but still with disease control problems. The progeny of a tebuconazole resistant (R) × sensitive (S) V. inaequalis cross were analysed in order to improve understanding of the genetic control of tebuconazole sensitivity. RESULTS: There is cross-resistance between myclobutanil and tebuconazole (r=0.91; P < 0.001). Sensitivity to tebuconazole of the progeny of a R×S cross varied quantitatively in a pattern which implied at least two gene loci differing between the parental strains. In addition, the asymmetric distribution of the sensitivity in the progeny implied possible epistatic effects. CONCLUSION: Resistance to myclobutanil and tebuconazole is strongly correlated. At least two genes are involved in the control of tebuconazole resistance in V. inaequalis

Integrating the Genetic and Physical Maps of Arabidopsis thaliana: Identification of Mapped Alleles of Cloned Essential (EMB) Genes

The classical genetic map of Arabidopsis includes more than 130 genes with an embryo-defective (emb) mutant phenotype. Many of these essential genes remain to be cloned. Hundreds of additional EMB genes have been cloned and catalogued (www.seedgenes.org) but not mapped. To facilitate EMB gene identification and assess the current level of saturation, we updated the classical map, compared the physical and genetic locations of mapped loci, and performed allelism tests between mapped (but not cloned) and cloned (but not mapped) emb mutants with similar chromosome locations. Two hundred pairwise combinations of genes located on chromosomes 1 and 5 were tested and more than 1100 total crosses were screened. Sixteen of 51 mapped emb mutants examined were found to be disrupted in a known EMB gene. Alleles of a wide range of published EMB genes (YDA, GLA1, TIL1, AtASP38, AtDEK1, EMB506, DG1, OEP80) were discovered. Two EMS mutants isolated 30 years ago, T-DNA mutants with complex insertion sites, and a mutant with an atypical, embryo-specific phenotype were resolved. The frequency of allelism encountered was consistent with past estimates of 500 to 1000 EMB loci. New EMB genes identified among mapped T-DNA insertion mutants included CHC1, which is required for chromatin remodeling, and SHS1/AtBT1, which encodes a plastidial nucleotide transporter similar to the maize Brittle1 protein required for normal endosperm development. Two classical genetic markers (PY, ALB1) were identified based on similar map locations of known genes required for thiamine (THIC) and chlorophyll (PDE166) biosynthesis. The alignment of genetic and physical maps presented here should facilitate the continued analysis of essential genes in Arabidopsis and further characterization of a broad spectrum of mutant phenotypes in a model plant

Evolving cell models for systems and synthetic biology

This paper proposes a new methodology for the automated design of cell models for systems and synthetic biology. Our modelling framework is based on P systems, a discrete, stochastic and modular formal modelling language. The automated design of biological models comprising the optimization of the model structure and its stochastic kinetic constants is performed using an evolutionary algorithm. The evolutionary algorithm evolves model structures by combining different modules taken from a predefined module library and then it fine-tunes the associated stochastic kinetic constants. We investigate four alternative objective functions for the fitness calculation within the evolutionary algorithm: (1) equally weighted sum method, (2) normalization method, (3) randomly weighted sum method, and (4) equally weighted product method. The effectiveness of the methodology is tested on four case studies of increasing complexity including negative and positive autoregulation as well as two gene networks implementing a pulse generator and a bandwidth detector. We provide a systematic analysis of the evolutionary algorithm’s results as well as of the resulting evolved cell models