Effects of NaCl on the response of <Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> callus to <Emphasis Type="Italic">Botrytis cinerea</Emphasis> infection

Callus of the halophyte Mesembryanthemum crystallinum was used to study the effect of NaCl on the response to Botrytis cinerea infection. The fungus easily colonized the callus surface and the intercellular spaces. However, in the NaCl-adapted tissues the incidence of penetration was 67 % lower than in the inoculated control tissue. The modification of the infection pattern found in the salt-adapted callus could be related to metabolic adaptations to salinity. This was manifested by the enhanced antioxidant potential of ascorbate, the up-regulated activities of ascorbate peroxidase, as well as guaiacol and syringaldazine peroxidases together with the increased detoxification capacity of glutathione transferase in the NaCl-adapted callus. The post-inoculation changes in NaCl-adapted and non-adapted calli were roughly similar and supported the prooxidative nature of B. cinerea infection.     

Selected reactive oxygen species and antioxidant enzymes in common bean after <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">phaseolicola</Emphasis> and <Emphasis Type="Italic">Botrytis cinerea</Emphasis> infection

Phaseolus vulgaris cv. Korona plants were inoculated with the bacteria Pseudomonas syringae pv. phaseolicola (Psp), necrotrophic fungus Botrytis cinerea (Bc) or with both pathogens sequentially. The aim of the experiment was to determine how plants cope with multiple infection with pathogens having different attack strategy. Possible suppression of the non-specific infection with the necrotrophic fungus Bc by earlier Psp inoculation was examined. Concentration of reactive oxygen species (ROS), such as superoxide anion (O₂ ^?) and H₂O₂ and activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were determined 6, 12, 24 and 48 h after inoculation. The measurements were done for ROS cytosolic fraction and enzymatic cytosolic or apoplastic fraction. Infection with Psp caused significant increase in ROS levels since the beginning of experiment. Activity of the apoplastic enzymes also increased remarkably at the beginning of experiment in contrast to the cytosolic ones. Cytosolic SOD and guaiacol peroxidase (GPOD) activities achieved the maximum values 48 h after treatment. Additional forms of the examined enzymes after specific Psp infection were identified; however, they were not present after single Bc inoculation. Subsequent Bc infection resulted only in changes of H₂O₂ and SOD that occurred to be especially important during plant–pathogen interaction. Cultivar Korona of common bean is considered to be resistant to Psp and mobilises its system upon infection with these bacteria. We put forward a hypothesis that the extent of defence reaction was so great that subsequent infection did not trigger significant additional response.     

<Emphasis Type="Italic">Erwinia carotovora</Emphasis> elicitors and <Emphasis Type="Italic">Botrytis cinerea</Emphasis> activate defense responses in <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Background  

Vascular plants respond to pathogens by activating a diverse array of defense mechanisms. Studies with these plants have provided a wealth of information on pathogen recognition, signal transduction and the activation of defense responses. However, very little is known about the infection and defense responses of the bryophyte, Physcomitrella patens, to well-studied phytopathogens. The purpose of this study was to determine: i) whether two representative broad host range pathogens, Erwinia carotovora ssp. carotovora (E.c. carotovora) and Botrytis cinerea (B. cinerea), could infect Physcomitrella, and ii) whether B. cinerea, elicitors of a harpin (HrpN) producing E.c. carotovora strain (SCC1) or a HrpN-negative strain (SCC3193), could cause disease symptoms and induce defense responses in Physcomitrella.     

Drill-assisted genomic DNA extraction from <Emphasis Type="Italic">Botrytis cinerea</Emphasis>

Current DNA extraction protocols for genomic DNA from Botrytis cinerea almost always start with mycelium that has been reduced to powder with liquid N₂ in a mortar, and this makes their application to a large number of samples slow and cumbersome. Here we present an adaptation of an existing method [Möller et al. (1992) Nucleic Acids Res 20: 6115–6116] for which the initial steps have been modified, including the homogenization of the fungus with sand and the aid of a common household drill. This method allows the processing of large number of samples in much shorter times and generates an average of 4 μg DNA per sample, of sufficient quality for use in PCR and Southern blotting.     

Three QTLs for <Emphasis Type="Italic">Botrytis cinerea</Emphasis> resistance in tomato

Tomato (Solanum lycopersicum) is susceptible to grey mold (Botrytis cinerea). Partial resistance to this fungus was identified in accessions of wild relatives of tomato such as S. habrochaites LYC4. In order to identify loci involved in quantitative resistance (QTLs) to B. cinerea, a population of 174 F₂ plants was made originating from a cross between S. lycopersicum cv. Moneymaker and S. habrochaites LYC4. The population was genotyped and tested for susceptibility to grey mold using a stem bioassay. Rbcq1, a QTL reducing lesion growth (LG) and Rbcq2, a QTL reducing disease incidence (DI) were identified. Rbcq1 is located on Chromosome 1 and explained 12% of the total phenotypic variation while Rbcq2 is located on Chromosome 2 and explained 15% of the total phenotypic variation. Both QTL effects were confirmed by assessing disease resistance in two BC₂S₁ progenies segregating for either of the two QTLs. One additional QTL, Rbcq4 on Chromosome 4 reducing DI, was identified in one of the BC₂S₁ progenies. F₂ individuals, homozygous for the Rbcq2 and Rbcq4 alleles of S. habrochaites showed a reduction of DI by 48%. QTLs from S. habrochaites LYC4 offer good perspectives for breeding B. cinerea resistant tomato cultivars. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

<Emphasis Type="Italic">Botrytis cinerea</Emphasis>-resistant marker-free <Emphasis Type="Italic">Petunia hybrida</Emphasis> produced using the MAT vector system

The presence of marker genes conferring antibiotic or herbicide resistance in transgenic plants has been a controversial issue and a serious problem for their public acceptance and commercialization. The MAT (multi-auto-transformation) vector system has been one of the strategies developed to excise the selection marker gene and produce marker-free transgenic plants. In an attempt to produce transgenic marker-free Petunia hybrida plants resistant to Botrytis cinerea (gray mold), we used the ipt gene as a selectable marker gene and the wasabi defensin (WD) gene, isolated from Wasabia japonica (a Japanese horseradish which has been a potential source of antimicrobial proteins), as a gene of interest. The WD gene was cloned from the binary vector, pEKH-WD, to an ipt-type MAT vector, pMAT21, by gateway cloning technology and transferred to Agrobacterium tumefaciens strain EHA105. Infected leaf explants of P. hybrida were cultured on hormone- and antibiotic-free MS medium. Extreme shooty phenotype (ESP)/ipt shoots were produced by the explants infected with the pMAT21-WD. The same antibiotic- and hormone-free MS medium was used in subcultures of the ipt shoots. Ipt shoots subsequently produced morphologically normal shoots. Molecular analyses of genomic DNA from the transgenic plants confirmed the integration of the gene of interest and excision of the selection marker. Expression of the WD gene was confirmed by northern blot and western blot analyses. A disease resistance assay of the marker-free transgenic plants exhibited enhanced resistance against B. cinerea strain 40 isolated from P. hybrida.     

Mapping of loci from <Emphasis Type="Italic">Solanum lycopersicoides</Emphasis> conferring resistance or susceptibility to <Emphasis Type="Italic">Botrytis cinerea</Emphasis> in tomato

Cultivated tomato (Solanum lycopersicum, syn. Lycopersicon esculentum) is susceptible to the necrotrophic ascomycete and causal agent of gray mold, Botrytis cinerea. Resistance to this fungal pathogen is elevated in wild relatives of tomato, including Solanum lycopersicoides. An introgression line population (IL) containing chromosomal segments of S. lycopersicoides within the background of tomato cv. VF36 was used to screen the genome for foliar resistance and susceptibility to B. cinerea. Based on this screen, putative quantitative trait loci (QTL) were identified, five for resistance and two for susceptibility. Four resistance QTL decreased infection frequency while the fifth reduced lesion diameter. One susceptibility QTL increased infection frequency whereas the other increased lesion diameter. Overlapping chromosomal segments provided strong evidence for partial resistance on chromosomes 1 and 9 and for elevated susceptibility on chromosome 11. Segregation analysis confirmed the major resistance QTL on the long arm of chromosome 1 and susceptibility on chromosome 11. Linkage of partial resistance to chromosome 9 could not be confirmed. The usefulness of these data for resistance breeding and for map-based cloning of foliar resistance to B. cinerea is discussed.     

Ultrastructure of ferritin in the leaves of <Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> under stress conditions

The location and structure of ferritin in the parenchyma of leaf minor veins of the common ice plant (Mesembryanthemum crystallinum L.) treated with exogenous putrescine under salinity conditions were investigated by electron microscopy. Considerable aggregates of ferritin were detected in the chloroplasts of bundle sheath cells, in companion phloem cells, and other parenchyma cells of leaf minor veins. The structure of ferritin in the vascular parenchyma chloroplasts suggests that it was partially degraded and converted to phytosiderin. This point of view is based on indistinct structure of Fe-containing cores of ferritin molecules, break of distance between the cores, and their pronounced ability to aggregate and produce larger structures. Aggregation of Fe-containing cores apparently pointed to the destruction of ferritin protein envelope or its partial degradation. In a certain stage of ferritin destruction, electron-dense material and the structures resembling small vesicles appeared between the Fe-containing cores. Electron-dense inclusions, whose structure was similar to that of phytosiderin, were also detected in the vacuoles. Examination of the cross sections done without additional staining showed that the same as ferritin, phytosiderin in the chloroplasts and vacuoles was dark-colored against weakly colored cellular structures. In the vascular parenchyma of control plant leaves, the level of ferritin and phytosiderin was greater than in the mesophyll and much lower than in the plants simultaneously treated with NaCl and putrescine. In control material, iron cores of ferritin and phytosiderin were more light-colored and 2–3 times smaller in size than in the experimental treatment. Destruction of ferritin essentially did not occur in the mesophyll but was observed in the chloroplasts of bundle sheath cells on the border between the mesophyll and vascular bundle. The presence of much ferritin and phytosiderin on the border between the mesophyll and the vessels is accounted for by the fact that the vascular parenchyma is a buffer area that maintains a specific concentration of iron in the mesophyll of leaves and other parts of the plant. Within the cell, the role of such a buffer is performed by ferritin and vacuoles. Transformation of ferritin to insoluble hydrophobic phytosiderin is supposed to be an efficient way of withdrawing the excess of active iron from the cellular metabolism and therefore of relaxing oxidative stress. Ferritin and phytosiderin were detected not only in parenchyma cells of leaf minor veins but in sieve tubes as well. This suggests that iron may be transported within the plant as a component of protein complex.     

Cytokinin and Ethylene Affect Auxin Transport-Dependent Rhizogenesis in Hypocotyls of Common Ice Plant (<Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Hypocotyl explants of Mesembryanthemum crystallinum regenerated roots when cultured vertically with either the apical end (AE) or basal end (BE) in media containing indole-3-acetic acid (IAA). IAA alone induced roots regularly from the basal end of the explants, either from the cut surface immersed in the medium or from the opposite side. The inhibitors of auxin efflux carriers, α-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), inhibited rhizogenesis only from AE-cultured explants, indicating the role of polar auxin transport in root regeneration in this system. Cytokinin (zeatin, kinetin, BAP) added to auxin-containing medium reduced rhizogenesis from the explants maintained with BE and AE and additionally changed the IAA-induced pattern of rooting in AE-cultured explants by favoring rooting from the apical end and middle part of the hypocotyl with its concomitant reduction from the basal end. The addition of kinetin did not influence the content of IAA in the explants maintained with AE, suggesting that the cytokinin effect on root patterning was not dependent on auxin biosynthesis. Kinetin, however, strongly enhanced ethylene production. The importance of ethylene in regulating PAT-dependent rhizogenesis was tested by using an ethylene antagonist AgNO₃, an inhibitor of ethylene synthesis aminoethoxyvinylglycine (AVG), and a precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC). AgNO₃ applied together with IAA or with IAA and kinetin strongly reduced the production of ethylene, inhibited rhizogenesis, and induced nonregenerative callus from BE, suggesting the need for ethylene signaling to elicit the rhizogenic action of auxin. A reduction of rhizogenesis and decrease of ethylene biosynthesis was also caused by AVG. In addition, AVG at 10 μM reversed the effect of cytokinin on root patterning, resulting in roots emerging only from BE on the medium with IAA and kinetin. Conversely, ACC at 200 μM markedly enhanced the production of ethylene and partly mimicked the effect of cytokinin when applied with IAA alone, thus confirming that in cultured hypocotyls of ice plant, cytokinin affects IAA-induced rhizogenesis through an ethylene-dependent pathway.     

Biological control of tomato gray mold caused by <Emphasis Type="Italic">Botrytis cinerea</Emphasis> by using <Emphasis Type="Italic">Streptomyces</Emphasis> spp.

Streptomyces is a genus known for its ability to protect plants against many pathogens and various strains of this bacteria have been used as biological control agents. In this study, the efficacy of Streptomyces philanthi RM-1-138, S. philanthi RL-1-178, and Streptomyce mycarofaciens SS-2-243 to control various strains of Botrytis cinerea was evaluated both in vitro and in vivo. In vitro studies using confrontation tests on PDA plates indicated that the three strains of Streptomyces spp. inhibited the growth of 41 strains of B. cinerea. Volatile compounds produced by Streptomyces spp. had an influence on the growth of ten strains of B. cinerea while its culture filtrate at low concentration (diluted at 10^?3) showed a complete inhibition (100%) of spore germination of B. cinerea strain BC1. A significant protection efficacy of tomato against B. cinerea was observed on both whole plant test (57.4%) and detached leaf test (60.1%) with S. philanti RM-1-138. Moreover, this antagonistic strain had a preventive and a curative effect. These results indicated that S. philanthi RM-1-138 may have the potential to control gray mold caused by B. cinerea on tomato but further work is required to enhance its efficacy and its survival in planta.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Chitosan improves development,and protects<Emphasis Type="Italic"> Vitis vinifera</Emphasis> L. against<Emphasis Type="Italic"> Botrytis cinerea</Emphasis>

We evaluated the potential of chitosan both to stimulate plant development and to induce protection from Botrytis cinerea in Vitis vinifera L. plantlets. The presence of 1.75% (v/v) chitogel in the culture medium was the optimal concentration for in vitro grapevine plantlet growth, as determined by measurements on enhancement of root and shoot biomass. Photosynthesis and related parameters were also stimulated in chitogel-treated plantlets. Chitogel reduced the development of Botrytis cinerea and induced cytological alterations to the pathogen. When challenged with the fungus, a significant decrease in disease incidence was observed in plants growing on medium supplemented with chitogel. Furthermore, exogenous foliar applications of chitogel to plantlets growing on chitogel-free medium sensitized them so as to be protected against Botrytis cinerea attack. Our results indicate that chitogel can be used in the vineyard as a means to attain protection against Botrytis cinerea and that its application may counteract the wide use of chemical pesticides.Communicated by S. Gleddie     

High-level overproduction of <Emphasis Type="Italic">Thermus</Emphasis> enzymes in <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Biotechnology needs to explore the capacity of different organisms to overproduce proteins of interest at low cost. In this paper, we show that Streptomyces lividans is a suitable host for the expression of Thermus thermophilus genes and report the overproduction of the corresponding proteins. This capacity was corroborated after cloning the genes corresponding to an alkaline phosphatase (a periplasmic enzyme in T. thermophilus) and that corresponding to a beta-glycosidase (an intracellular enzyme) in Escherichia coli and in S. lividans. Comparison of the production in both hosts revealed that the expression of active protein achieved in S. lividans was much higher than in E. coli, especially in the case of the periplasmic enzyme. In fact, the native signal peptide of the T. thermophilus phosphatase was functional in S. lividans, being processed at the same peptide bond in both organisms, allowing the overproduction and secretion of this protein to the S. lividans culture supernatant. As in E. coli, the thermostability of the expressed proteins allowed a huge purification factor upon thermal denaturation and precipitation of the host proteins. We conclude that S. lividans is a very efficient and industry-friendly host for the expression of thermophilic proteins from Thermus spp.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.