Description of Siccibacter colletis sp. nov., a novel species isolated from plant material, and emended description of Siccibacter turicensis.

A re-evaluation of the taxonomic position of two strains, 1383(T) and 2249, isolated from poppy seeds and tea leaves, which had been identified as Siccibacter turicensis (formerly Cronobacter zurichensis ), was carried out. The analysis included phenotypic characterization, 16S rRNA gene sequencing, multilocus sequence analysis (MLSA) of five housekeeping genes (atpD, fusA, glnS, gyrB and infB; 2034 bp) and ribosomal MLSA (53 loci; 22 511 bp). 16S rRNA gene sequence analysis and MLSA showed that the strains formed an independent phylogenetic lineage, with Siccibacter turicensis LMG 23730(T) as the closest neighbour. Average nucleotide identity analysis and phenotypic analysis confirmed that these strains represent a novel species, for which the name Siccibacter colletis sp. nov. is proposed. The type strain is 1383(T) ( = NCTC 14934(T) = CECT 8567(T) = LMG 28204(T)). An emended description of Siccibacter turicensis is also provided.

Disentangling Peronospora on Papaver: phylogenetics, taxonomy, nomenclature and host range of downy mildew of opium poppy (Papaver somniferum) and related species.

Based on sequence data from ITS rDNA, cox1 and cox2, six Peronospora species are recognised as phylogenetically distinct on various Papaver species. The host ranges of the four already described species P. arborescens, P. argemones, P. cristata and P. meconopsidis are clarified. Based on sequence data and morphology, two new species, P. apula and P. somniferi, are described from Papaver apulum and P. somniferum, respectively. The second Peronospora species parasitizing Papaver somniferum, that was only recently recorded as Peronospora cristata from Tasmania, is shown to represent a distinct taxon, P. meconopsidis, originally described from Meconopsis cambrica. It is shown that P. meconopsidis on Papaver somniferum is also present and widespread in Europe and Asia, but has been overlooked due to confusion with P. somniferi and due to less prominent, localized disease symptoms. Oospores are reported for the first time for P. meconopsidis from Asian collections on Papaver somniferum. Morphological descriptions, illustrations and a key are provided for all described Peronospora species on Papaver. cox1 and cox2 sequence data are confirmed as equally good barcoding loci for reliable Peronospora species identification, whereas ITS rDNA does sometimes not resolve species boundaries. Molecular phylogenetic data reveal high host specificity of Peronospora on Papaver, which has the important phytopathological implication that wild Papaver spp. cannot play any role as primary inoculum source for downy mildew epidemics in cultivated opium poppy crops.

Enhanced morphinan alkaloid production in hairy root cultures of Papaver bracteatum by over-expression of salutaridinol 7-o-acetyltransferase gene via Agrobacterium rhizogenes mediated transformation.

Papaver bracteatum is an important medicinal plant valued for its high content of thebaine and an alternative to P. somniferum for benzylisoquinoline alkaloid production. Salutaridinol 7-o-acetyltransferase (SalAT) is a key gene in morphinan alkaloids biosynthesis pathway. Over expression of SalAT gene was used for metabolic engineering in P. bracteatum hairy root cultures. Transcript level of the salutaridinol 7-o-acetyltransferase gene in transgenic hairy root lines increased up to 154 and 128 % in comparison with hairy roots without SalAT over expression and wild type roots, respectively. High performance liquid chromatography analysis showed that the transgenic hairy roots relatively improved levels of thebaine (1.28 % dry weight), codeine (0.02 % dry weight) and morphine (0.03 % dry weight) compared to those hairy roots without SalAT over expression. This suggests that P. bracteatum hairy roots expressing the SalAT gene could be potentially used for the production of valuable morphinan alkaloids.

Systemic protection of Papaver somniferum L. against Iraella luteipes (Hymenoptera: Cynipidae) by an endophytic strain of Beauveria bassiana (Ascomycota: Hypocreales).

The poppy stem gall wasp, Iraella luteipes (Thompson) (Hymenoptera: Cynipidae), is one of the main pests of the opium poppy, Papaver somniferum L., an economically important pharmaceutical crop cultivated worldwide. In a previous study, we obtained from I. luteipes larvae a strain of the entomopathogenic fungus Beauveria bassiana (Ascomycota: Hypocreales) that can become established endophytically in opium poppy plants. A field experiment was conducted to study the ability of this B. bassiana strain to provide systemic protection against damage by I. luteipes in opium poppy in southern Spain for three seasons. Conidial suspensions were applied as seed dressings, leaf sprays, or soil sprays. The effect of the treatment was studied by harvesting fully ripened plants and dissecting I. luteipes larvae from the stem. The effect of treatment on growth and yield was also evaluated. Emergence of I. luteipes adults was not uniform over the 3 yr, with important differences exhibited in the duration of the emergence period, although the flight peaks tended to occur in mid-late April. B. bassiana seed dressings, leaf sprays at the fourth true-leaf stage, and soil sprays were not significantly different in their ability to reduce the number of larvae per plant compared with the controls, with percentage reductions of 36.5-58.5, 64.4-73.4, and 51.9-57.2% in 2005, 2006, and 2007, respectively. Even though the population level of I. luteipes increased over the 3 yr, the efficacy of the fungal inoculation in reducing the larval population was maintained throughout the study period. No significant differences between inoculation methods were detected in the percentage of leaf pieces showing fungal growth when placed on B. bassiana selective medium, with mean values in the range of 10-15% for the three seasons. Leaf pieces from controls did not exhibit any sign of B. bassiana growth when placed on B. bassiana-selective medium. Neither adverse effects on growth and yield nor symptomatic tissues were observed in B. bassiana-treated plants compared with controls in any of the three seasons.

Quantitative 1H NMR metabolomics reveals extensive metabolic reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures.

BACKGROUND: Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a model system to study plant alkaloid metabolism. The plant is cultivated as the only commercial source of the narcotic analgesics morphine and codeine, but also produces many other alkaloids including the antimicrobial agent sanguinarine. Modulations in plant secondary metabolism as a result of environmental perturbations are often associated with the altered regulation of other metabolic pathways. As a key component of our functional genomics platform for opium poppy we have used proton nuclear magnetic resonance (1H NMR) metabolomics to investigate the interplay between primary and secondary metabolism in cultured opium poppy cells treated with a fungal elicitor. RESULTS: Metabolite fingerprinting and compound-specific profiling showed the extensive reprogramming of primary metabolic pathways in association with the induction of alkaloid biosynthesis in response to elicitor treatment. Using Chenomx NMR Suite v. 4.6, a software package capable of identifying and quantifying individual compounds based on their respective signature spectra, the levels of 42 diverse metabolites were monitored over a 100-hour time course in control and elicitor-treated opium poppy cell cultures. Overall, detectable and dynamic changes in the metabolome of elicitor-treated cells, especially in cellular pools of carbohydrates, organic acids and non-protein amino acids were detected within 5 hours after elicitor treatment. The metabolome of control cultures also showed substantial modulations 80 hours after the start of the time course, particularly in the levels of amino acids and phospholipid pathway intermediates. Specific flux modulations were detected throughout primary metabolism, including glycolysis, the tricarboxylic acid cycle, nitrogen assimilation, phospholipid/fatty acid synthesis and the shikimate pathway, all of which generate secondary metabolic precursors. CONCLUSION: The response of cell cultures to elicitor treatment involves the extensive reprogramming of primary and secondary metabolism, and associated cofactor biosynthetic pathways. A high-resolution map of the extensive reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures is provided.

Genetic parameters and correlations of collar rot resistance with important biochemical and yield traits in opium poppy (Papaver somniferum L.).

Collar rot, caused by Rhizoctonia solani Kühn, is one of the most severe fungal diseases of opium poppy. In this study, heritability, genetic advance and correlation for 10 agronomic, 1 physiological, 3 biochemical and 1 chemical traits with disease severity index (DSI) for collar rot were assessed in 35 accessions of opium poppy. Most of the economically important characters, like seed and capsule straw yield per plant, oil and protein content of seeds, peroxidase activity in leaves, morphine content of capsule straw and DSI for collar rot showed high heritability as well as genetic advance. Highly significant negative correlation between DSI and seed yield clearly shows that as the disease progresses in plants, seed yield declines, chiefly due to premature death of infected plants as well as low seed and capsule setting in the survived population of susceptible plants. Similarly, a highly significant negative correlation between peroxidase activity and DSI indicated that marker-assisted selection of disease-resistant plants based on high peroxidase activity would be effective and survived susceptible plants could be removed from the population to stop further spread.

[Study on the pathogen and its biological characteristics of opium poppy downy mildew].

OBJECTIVE: To study the pathogen of opium poppy downy mildew and its biological characteristic for further research on the disease. METHOD: Development of the disease was observed systematically in the field. Germination rate of sporangium in different temperature, pH and nutrition was examined with suspending-drop method. Slide-germination method was used to observe its germination in different humidity maintained by different concentration of H2SO4. RESULT AND CONCLUSION: The disease manifests itself in two forms: severely infected plants (systematic infection) and leaf spots (nonsystematic infection). Sporangia of the pathogen are oval or globular, thin walled, smooth, hyaline, with 7.74-16.34 microns diameter in base 1 and 8.34-15.05 microns in base 2.0 ospores are light yellow with 33.87-70.54 microns x 19.34-62.64 microns in base 1 and 36.85-49.68 microns x 42.08-55.76 microns in base 2. Conidiophores are stout, erect, whose branching times and length are different between those in base 1 and those in base 2. Sporangia sprot directly in two hours. Film of water is necessary for sporangium to sprot. The optimum temperature range of sporangium sprot is 12-21 degrees C, the best being 16 degrees C, the pH range is 4.53-9.18 the best optimum at pH 7.38, and the extract of leaf of 1:5 is good for its germination.

[Field test and lab experiment on control efficacy of the pathogen of opium poppy mildew].

OBJECTIVE: To screen effectual fungicides for field control because of the seriousness of opium poppy mildew and importance of chemical control on plant diseases. METHOD: Seven fungicides were screened in Lab experiment and field test during 1996-1997. RESULT AND CONCLUSION: All of them and their different dosages were effective to control conidia of Peronospora arborescens. Among them, 72.2% propamocarb of 1203 and 902.5 ppm were the most effective both in Lab experiment and field test with efficacy 79.91% and 79.33% respectively in field test, and the efficacy of other fungicides was over 50%. Seven fungicides tested can be used to control nonsystematic symptom of opium poppy mildew.

Uncoupled defense gene expression and antimicrobial alkaloid accumulation in elicited opium poppy cell cultures.

Treatment of opium poppy (Papaver somniferum L.) cell cultures with autoclaved mycelial homogenates of Botrytis sp. resulted in the accumulation of sanguinarine. Elicitor treatment also caused a rapid and transient induction in the activity of tyrosine/dopa decarboxylase (TYDC, EC 4.1.1.25), which catalyzes the conversion of L-tyrosine and L-dopa to tyramine and dopamine, respectively, the first steps in sanguinarine biosynthesis. TYDC genes were differentially expressed in response to elicitor treatment. TYDC1-like mRNA levels were induced rapidly but declined to near baseline levels within 5 h. In contrast, TYDC2-like transcript levels increased more slowly but were sustained for an extended period. Induction of TYDC mRNAs preceded that of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) mRNAs. An elicitor preparation from Pythium aphanidermatum was less effective in the induction of TYDC mRNA levels and alkaloid accumulation; however, both elicitors equally induced accumulation of PAL transcripts. In contrast, treatment with methyl jasmonate resulted in an induction of TYDC but not PAL mRNAs. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and the protein kinase inhibitor staurosporine partially blocked the fungal elicitor-induced accumulation of sanguinarine. However, only staurosporine and okadaic acid, an inhibitor of protein phosphatases 1 and 2A, blocked the induction of TYDC1-like transcript levels, but they did not block the induction of TYDC2-like or PAL transcript levels. These data suggest that activation mechanisms for PAL, TYDC, and some later sanguinarine biosynthetic enzymes are uncoupled.