Ewa Łojkowska

Genotyping of bacteria belonging to the former Erwinia genus by PCR-RFLP analysis of a recA gene fragment.

Genotypic characterization, based on the analysis of restriction fragment length polymorphism of the recA gene fragment PCR product (recA PCR-RFLP), was performed on members of the former Erwinia genus. PCR primers deduced from published recA gene sequences of Erwinia carotovora allowed the amplification of an approximately 730 bp DNA fragment from each of the 19 Erwinia species tested. Amplified recA fragments were compared using RFLP analysis with four endonucleases (AluI, HinfI, TasI and Tru1I), allowing the detection of characteristic patterns of RFLP products for most of the Erwinia species. Between one and three specific RFLP groups were identified among most of the species tested (Erwinia amylovora, Erwinia ananas, Erwinia cacticida, Erwinia cypripedii, Erwinia herbicola, Erwinia mallotivora, Erwinia milletiae, Erwinia nigrifluens, Erwinia persicina, Erwinia psidii, Erwinia quercina, Erwinia rhapontici, Erwinia rubrifaciens, Erwinia salicis, Erwinia stewartii, Erwinia tracheiphila, Erwinia uredovora, Erwinia carotovora subsp. atroseptica, Erwinia carotovora subsp. betavasculorum, Erwinia carotovora subsp. odorifera and Erwinia carotovora subsp. wasabiae). However, in two cases, Erwinia chrysanthemi and Erwinia carotovora subsp. carotovora, 15 and 18 specific RFLP groups were detected, respectively. The variability of genetic patterns within these bacteria could be explained in terms of their geographic origin and/or wide host-range. The results indicated that PCR-RFLP analysis of the recA gene fragment is a useful tool for identification of species and subspecies belonging to the former Erwinia genus, as well as for differentiation of strains within E. carotovora subsp. carotovora and E. chrysanthemi.

ESTABLISHMENT OF HAIRY ROOT CULTURES OF AMMI MAJUS

Axenically grown Ammi majus plantlets were inoculated with seven different Agrobacterium rhizogenes strains. Hairy root lines were established only after inoculation with the two agropine strains: A4 and LBA9402. The growth rate of hairy root cultures was about thirty times faster than that of callus and cell suspension cultures. Polymerase chain reaction with primers for the genes rolB and rolC confirmed the integration of the T-DNA fragment of Ri plasmid of A. rhizogenes to the genome of hairy roots obtained after transformation by both Agrobacterium strains. The furanocoumarins (psoralen, xanthotoxine, bergapten and imperatorin) usually found in seeds of A. majus were not detected in callus, cell suspension and hairy root cultures using Gas chromatography-mass spectrometry (GC-MS). However, umbelliferone, a precursor of furanocoumarins, was detected in callus, cell suspension and hairy root cultures. The umbelliferone content in extracts of hairy root cultures, obtained after transformation by A4, was similar to that determined in A. majus seeds (19 µg/g DW) and higher than those obtained for cell suspension and callus cultures (2 and 9 µg/g DW, respectively).

Elicitation of secondary metabolites in in vitro cultures of Ammi majus L.

Abstract The present study was concentrated on the production of secondary metabolites in callus, cell suspension and hairy roots of Ammi majus L. by exposing them to elicitors: benzo(1,2,3)-thiadiazole-7-carbothionic acid S-methyl ester (BION®) and autoclaved lysate of cell suspension of bacteria—Enterobacter sakazaki. GC and GC–MS analysis of chloroform and methanol extracts indicated a higher accumulation of umbelliferone in the elicited tissues than in the control ones. Using GC–MS, two compounds not earlier found in A. majus tissues were identified in callus cultures: scopoletin (7-hydroxy-6-metoxy-2H-1-benzopyron-2-one) and dehydrogeijerin (7-methoxy-6-(3-methyl-1-oxo-2-butenyl)-2H-1-benzopyran-2-one).

Application of chitin and chitosan as elicitors of coumarins and furoquinolone alkaloids in Ruta graveolens L. (common rue)

Common rue (Ruta gra veolens L.) accumulates various types of secondary metabolites, such as coumarins furanocoumarins, acridone and quinolone alkaloids and flavonoids. Elicitation is a tool extensively used for enhancing secondary‐metabolite yields. Chitin and chitosan are examples of elicitors inducing phytoalexin accumulation in plant tissue. The present paper describes the application of chitin and chitosan as potential elicitors of secondary‐metabolite accumulation in R. graveolens shoots cultivated in vitro . The simple coumarins, linear furanocoumarins, dihydrofuranocoumarins and furoquinolone alkaloids biosynthesized in the presence of chitin and chitosan were isolated, separated and identified. There was a significant increase in the growth rate of R. graveolens shoots in the presence of either chitin or chitosan. Moreover, the results of the elicitation of coumarins and alkaloids accumulated by R. graveolens shoots in the presence of chitin and chitosan show that both compounds induced a significant increase in the concentrations of nearly all the metabolites. Adding 0.01% chitin caused the increase in the quantity (μg/g dry weight) of coumarins (pinnarin up to 116.7, rutacultin up to 287.0, bergapten up to 904.3, isopimpinelin up to 490.0, psoralen up to 522.2, xanhotoxin up to 1531.5 and rutamarin up to 133.7). The higher concentration of chitosan (0.1%) induced production of simple coumarins (pinnarin up to 116.7 and rutacultin up to 287.0), furanocoumarins (bergapten up to 904.3, isopimpinelin up to 490.0, psoralen up to 522.2, xanhotoxin up to 1531.5) and dihydrofuranocoumarins (chalepin up to 18 and rutamarin up to 133.7). Such a dramatic increase in the production of nearly all metabolites suggests that these compounds may be participating in the natural resistance mechanisms of R. graveolens . The application of chitin‐ and chitosan‐containing media may be considered a promising prospect in the biotechnological production of xanthotoxin, isopimpinelin, psoralen, chalepin or methoxylated dictamnine derivatives.

Chromatographic analysis of simple phenols in some species from the genus Salix

INTRODUCTION Salicis Cortex, made from willow bark is a herbal remedy, which is standardised based on the content of salicin, a compound with analgesic and antiphlogistic properties. However, clinical trials suggest that other compounds also present in Salicis Cortex can contribute to the pharmacological effects. OBJECTIVE To characterise the composition of phenolic acids in the barks of different species and clones from the genus Salix by use of chromatographic methods--HPTLC and HPLC. METHODOLOGY The phenolic acid composition was analysed by MGD (multiple gradient development)-HPTLC technique. The separation was performed on HPTLC Diol plates with gradient elution using a mixture of chloroform:hexane:ethyl acetate with increasing concentration of ethyl acetate from 10 to 25%. Derivatisation with thymol reagent was employed for the first time for specific detection of phenolic acids containing methoxyl groups. RESULTS The presence of all phenolic acids previously reported in the genus Salix was confirmed, namely p-hydroxybenzoic, vanillic, cinnamic, p-coumaric, ferulic and caffeic acids. Furthermore, pyrocatechol as a constituent of willow bark was revealed. The highest concentration of this compound was observed in the S. purpurea bark (2.25 mg/g). CONCLUSION The presence of a relatively high content of pyrocatechol in Salix species may raise doubts about the safe application of this herbal medicine.

Chemical structure of the O-polysaccharide isolated from Pectobacterium atrosepticum SCRI 1039.

The lipopolysaccharide (LPS) of the bacterium Pectobacterium atrosepticum SCRI 1039 was hydrolyzed and the products were separated. A study of the obtained O-polysaccharide by means of chemical methods, GLC, GLC-MS, and NMR spectroscopy allowed us to identify a branched polymer with a pentasaccharide repeating unit of the structure shown below, in which the fucose residue was partially O-acetylated at C-2, C-3 or C-4.

The complete genome, structural proteome, comparative genomics and phylogenetic analysis of a broad host lytic bacteriophage ϕD3 infecting pectinolytic Dickeya spp.

Plant necrotrophic Dickeya spp. are among the top ten most devastating bacterial plant pathogens able to infect a number of different plant species worldwide including economically important crops. Little is known of the lytic bacteriophages infecting Dickeya spp. A broad host lytic bacteriophage ϕD3 belonging to the family Myoviridae and order Caudovirales has been isolated in our previous study. This report provides detailed information of its annotated genome, structural proteome and phylogenetic relationships with known lytic bacteriophages infecting species of the Enterobacteriaceae family.

Identification of a DNA restriction-modification system in Pectobacterium carotovorum strains isolated from Poland

Aims:  Polish isolates of pectinolytic bacteria from the species Pectobacterium carotovorum were screened for the presence of a DNA restriction–modification (R–M) system.