Differential interference with Pythium ultimum sporangial activation and germination by Enterobacter cloacae in the corn and cucumber spermospheres

Differential protection of plants by Enterobacter cloacae was studied by investigating early sensing and response behavior of Pythium ultimum sporangia toward seeds in the presence or absence of E. cloacae. Ten percent of P. ultimum sporangia were activated within the first 30 min of exposure to cucumber seeds. In contrast, 44% of the sporangia were activated as early as 15 min after exposure to corn seeds with over 80% sporangial activation by 30 min. Germ tubes emerged from sporangia after 2.5 and 1.0 h in the cucumber and corn spermospheres, respectively. Seed application of the wild-type strain of E. cloacae (EcCT-501R3) reduced sporangial activation by 45% in the cucumber spermosphere, whereas no reduction was observed in the corn spermosphere. Fatty acid transport and degradation mutants of E. cloacae (strains EcL1 and Ec31, respectively) did not reduce sporangial activation in either of the spermospheres. Although wild-type or mutant strains of E. cloacae failed to reduce seed colonization incidence, pathogen biomass on cucumber seeds was reduced in the presence of E. cloacae strains EcCT-501R3 and Ec31 by 4 and 8 h after sowing, respectively. By 12 h, levels of P. ultimum on cucumber seeds treated with E. cloacae EcCT-501R3 did not differ from levels on noninoculated seeds. On corn seeds, P. ultimum biomass was not affected by the presence of any E. cloacae strain. When introduced after sporangial activation had occurred, E. cloacae failed to reduce P. ultimum biomass on cucumber seeds compared with that on nontreated seeds. Also, increasing numbers of sporangia used to inoculate seeds yielded increased pathogen biomass at each sampling time. This indicates a direct link between the level of seed-colonizing biomass of P. ultimum and the number of activated and germinated sporangia in the spermosphere, suggesting that E. cloacae suppresses P. ultimum seed infections by reducing sporangial activation and germination within the first 30 to 90 min after sowing.     

Regulation of the germination of Aleppo pine (Pinus halepensis) by nitrate, ammonium, and gibberellin, and its role in post-fire forest regeneration

Aleppo pine ( Pinus halepensis Mill.) regenerates after wildfires only by germination. This post-fire germination is controlled by temperature, light and the chemical characteristics of the ash-bed. The extremely high pH caused by the ash inhibits germination of Aleppo pine as well as of many other species. Here we report that ash extracts at neutral pH stimulate P. halepensis germination. The effects of ash extracts, nitrate, ammonium (in concentrations present in the ash) and GA₃ on the germination of P. halepensis seeds and on lipase activity at early germination were studied under controlled conditions. Sub-optimal concentrations of nitrate- and ammonium-stimulated germination and the activity of lipase in the seeds. It is proposed that nitrate increases seed sensitivity to GA₃ while ammonium directly affects lipase activity. The balance between the inhibition of germination by the high pH of ash and its stimulation by the nitrate and ammonium present in the ash determines the overall effect of the ash on post-fire germination of Aleppo pine. The pine seedlings appear in high density among the trees but because of the effects of the ash they appear in sparse stands under the projection of the burned pine canopies. These seedlings benefit from weak inter- and intra-specific competition as well as improved mineral nutrition and have good chances to grow into large trees that will form the regenerating forest; therefore, the enhancement of P. halepensis germination by nitrate and ammonium and its inhibition by high pH can be considered as ecophysiological adaptations to post-fire regeneration.     

Fatty acid competition as a mechanism by which Enterobacter cloacae suppresses Pythium ultimum sporangium germination and damping-off

Interactions between plant-associated microorganisms play important roles in suppressing plant diseases and enhancing plant growth and development. While competition between plant-associated bacteria and plant pathogens has long been thought to be an important means of suppressing plant diseases microbiologically, unequivocal evidence supporting such a mechanism has been lacking. We present evidence here that competition for plant-derived unsaturated long-chain fatty acids between the biological control bacterium Enterobacter cloacae and the seed-rotting oomycete, Pythium ultimum, results in disease suppression. Since fatty acids from seeds and roots are required to elicit germination responses of P. ultimum, we generated mutants of E. cloacae to evaluate the role of E. cloacae fatty acid metabolism on the suppression of Pythium sporangium germination and subsequent plant infection. Two mutants of E. cloacae EcCT-501R3, Ec31 (fadB) and EcL1 (fadL), were reduced in beta-oxidation and fatty acid uptake, respectively. Both strains failed to metabolize linoleic acid, to inactivate the germination-stimulating activity of cottonseed exudate and linoleic acid, and to suppress Pythium seed rot in cotton seedling bioassays. Subclones containing fadBA or fadL complemented each of these phenotypes in Ec31 and EcL1, respectively. These data provide strong evidence for a competitive exclusion mechanism for the biological control of P. ultimum-incited seed infections by E. cloacae where E. cloacae prevents the germination of P. ultimum sporangia by the efficient metabolism of fatty acid components of seed exudate and thus prevents seed infections.     

Differential inactivation of seed exudate stimulation of Pythium ultimum sporangium germination by Enterobacter cloacae influences biological control efficacy on different plant species

This study was initiated to understand whether differential biological control efficacy of Enterobacter cloacae on various plant species is due to differences in the ability of E. cloacae to inactivate the stimulatory activity of seed exudates to Pythium ultimum sporangium germination. In biological control assays, E. cloacae was effective in controlling Pythium damping-off when placed on the seeds of carrot, cotton, cucumber, lettuce, radish, tomato, and wheat but failed to protect corn and pea from damping-off. Seeds from plants such as corn and pea had high rates of exudation, whereas cotton and cucumber seeds had much lower rates of exudation. Patterns of seed exudation and the release of P. ultimum sporangium germination stimulants varied among the plants tested. Seed exudates of plants such as carrot, corn, lettuce, pea, radish, and wheat were generally more stimulatory to P. ultimum than were the exudates of cotton, cucumber, sunflower, and tomato. However, this was not directly related to the ability of E. cloacae to inactivate the stimulatory activity of the exudate and reduce P. ultimum sporangium germination. In the spermosphere, E. cloacae readily reduced the stimulatory activity of seed exudates from all plant species except corn and pea. Our data have shown that the inability of E. cloacae to protect corn and pea seeds from Pythium damping-off is directly related to its ability to inactivate the stimulatory activity of seed exudates. On all other plants tested, E. cloacae was effective in suppressing damping-off and inactivating the stimulatory activity of seed exudates.     

Auxin and flavonoids in the progame phase of fertilization in petunia

The contents of IAA and flavonoids (Fls) were monitored in developing anthers, in vitro growing pollen tubes, and in the in vivo pollen-pistil system of two petunia (Petunia hybrida L.) clones, self-compatible and self-incompatible. In both clones, the development of male gametophytes was accompanied by the increase in the IAA (from 10 to 60–70 ng/g fr wt) and Fls (from 2 to 20 mg/g fr wt) contents. In both clones, pollen grain germination was accompanied by a substantial (by 10–30%) increase in the IAA content during the first two hours and Fl content during the first hour. Treatments with IAA and Fls stimulated both in vitro pollen grain germination and pollen tube growth by 25–30%. Male gametophyte germination in vivo, on the pistil surface, was accompanied by the increase in the IAA content from 90 to 200 ng/g fr wt during 8 h, whereas the content of Fl increased from 2 to 3 mg/g fr wt during the first hour and was maintained later at this level. In the pollen-pistil system, IAA and Fls were distributed evenly in the tissues of stigma, style, and ovary. On the basis of data obtained, we concluded that Fls might be endogenous mediators of IAA transport, which is one of the principal regulators of male gametophyte growth and development in the progame phase of fertilization, but are not involved in the mechanism of gametophyte incompatibility.     

Germination and <Emphasis Type="Italic">In Vitro</Emphasis> Growth of Petunia Male Gametophyte Are Affected by Exogenous Hormones and Involve the Changes in the Endogenous Hormone Level

The data obtained characterize the changes in the contents of endogenous phytohormones (IAA, cytokinins, GA, and ABA) in germinating pollen grains and growing pollen tubes of a self-compatible clone of petunia (sPetunia hybrida L.) within an 8-h period under in vitro conditions. The hydration and initiation of germination of pollen grains brought the ABA content down to a zero level, while the levels of GA, IAA, and cytokinins increased 1.5–2-fold. Later, in the growing pollen tubes, the GA content increased twofold, while the levels of IAA and cytokinins decreased. The exogenous ABA and GA₃ considerably promoted pollen germination and pollen tube growth; however, only the treatment with GA₃ produced the maximum length of pollen tubes. The exogenous IAA promoted and the exogenous cytokinins hindered the growth of pollen tubes. The membrane potential, as assessed with a potential-sensitive dye diS-C₃-(5), considerably increased in the pollen grains treated with ABA and benzyladenine, whereas IAA and GA₃ did not practically affect it. The authors conclude that the mature pollen grains contain the complete set of hormones essential for pollen germination and pollen tube growth. ABA, GA, and IAA together with cytokinins control the processes of pollen grain hydration, germination, and pollen tube growth, respectively.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 584–590.Original Russian Text Copyright © 2005 by Kovaleva, Zakharova, Minkina, Timofeeva, Andreev.     

Cloning and characterization of a locus encoding an indolepyruvate decarboxylase involved in indole-3-acetic acid synthesis in Erwinia herbicola.

Erwinia herbicola 299R synthesizes indole-3-acetic acid (IAA) primarily by the indole-3-pyruvic acid pathway. A gene involved in the biosynthesis of IAA was cloned from strain 299R. This gene (ipdC) conferred the synthesis of indole-3-acetaldehyde and tryptophol upon Escherichia coli DH5 alpha in cultures supplemented with L-tryptophan. The deduced amino acid sequence of the gene product has high similarity to that of the indolepyruvate decarboxylase of Enterobacter cloacae. Regions within pyruvate decarboxylases of various fungal and plant species also exhibited considerable homology to portions of this gene. This gene therefore presumably encodes an indolepyruvate decarboxylase (IpdC) which catalyzes the conversion of indole-3-pyruvic acid to indole-3-acetaldehyde. Insertions of Tn3-spice within ipdC abolished the ability of strain 299R to synthesize indole-3-acetaldehyde and tryptophol and reduced its IAA production in tryptophan-supplemented minimal medium by approximately 10-fold, thus providing genetic evidence for the role of the indolepyruvate pathway in IAA synthesis in this strain. An ipdC probe hybridized strongly with the genomic DNA of all E. herbicola strains tested in Southern hybridization studies, suggesting that the indolepyruvate pathway is common in this species. Maximum parsimony analysis revealed that the ipdC gene is highly conserved within this group and that strains of diverse geographic origin were very similar with respect to ipdC.     

Free IAA in stigmas and styles during pollen germination and pollen tube growth of Nicotiana tabacum

Although many studies have emphasized the importance of auxin in plant growth and development, the thorough understanding of its effect on pollen–pistil interactions is largely unknown. In this study, we investigated the role of free IAA in pollen–pistil interactions during pollen germination and tube growth in Nicotiana tabacum L. through using histo and subcellular immunolocalization with auxin monoclonal antibodies, quantification by HPLC and ELISA together with GUS staining in DR5::GUS -transformed plants. The results showed that free IAA in unpollinated styles was higher in the apical part and basal part than in the middle part, and it was more abundant in the transmitting tissue (TT). At the stage of pollen germination, IAA reached its highest content in the stigma and was mainly distributed in TT. After the pollen tubes entered the styles, the signal increased in the part where pollen tubes would enter and then rapidly declined in the part where pollen tubes had penetrated. Subcellular localization confirmed the presence of IAA in TT cells of stigmas and styles. Accordingly, a schematic diagram summarizes the changing pattern of free IAA level during flowering, pollination and pollen tube growth. Furthermore, we presented evidence that low concentration of exogenous IAA could, to a certain extent, facilitate in vitro pollen tube growth. These results suggest that IAA may be directly or indirectly involved in the pollen–pistil interactions. Additionally, some improvements of the IAA immunolocalization technique were made.     

Chromate-resistance in a chromate-reducing strain of Enterobacter cloacae

Resistance to toxic hexavalent chromium (chromate: CrO4(2)) in Enterobacter cloacae strain HO1, isolated from an activated sludge sample, was investigated under aerobic and anaerobic conditions. Decreased uptake of 51CrO4(2-) in E. cloacae strain HO1 was observed under aerobic conditions, when compared with a standard laboratory E. cloacae strain (IAM 1624). Under anaerobic conditions E. cloacae strain HO1 was able to reduce hexavalent chromium to the less toxic trivalent form. When E. clocacae strain HO1 was grown with nitrate anaerobically, the cells were observed to lose simultaneously their chromate-reducing ability and chromate-resistance under anaerobic conditions.     

Pollen tube growth is affected by exogenous hormones and correlated with hormone changes in styles in <Emphasis Type="Italic">Torenia fournieri</Emphasis> L.

In the present report, we described the effects of indole-3-acetic acid (IAA), zeatin (ZT), gibberellin (GA₃), and abscisic acid (ABA) on in vitro pollen germination and pollen tube growth in Torenia fournieri L. The results showed that IAA and GA₃ stimulated in vitro pollen tube growth, ABA inhibited pollen tube growth, and ZT had no significant effect on the process. The stimulating effect of exogenous IAA was particularly distinct, and led to synchronous growth of straighter and more slender pollen tubes compared with the controls. However, no significant changes were found in the germination of the treated pollen. The auxin efflux inhibitor, 10 μM 1-N-naphthylphthalamic acid (NPA), was also found to stimulate pollen tube growth. We measured the content of hormones (free IAA, ZT, GA₃, and ABA) in the stigmas and styles before and after pollination. The hormone contents of stigmas measured 0.5 h after pollination (0.5 HAP) showed that ABA content decreased, whereas the content of IAA, ZT, or GA₃ did not change significantly. The hormone level in pollinated styles (4 HAP) when pollen tubes had grown into the middle part of style was characterized by an increase in free IAA and GA₃ and a decrease in ABA, which was in agreement with the results that IAA and GA₃ promoted but ABA inhibited pollen tube growth in vitro. Furthermore, the change of IAA level in styles was most notable, which was accordant to the fact that auxin stimulated significantly pollen tube growth in vitro. Using immunoenzyme and immunogold labeling techniques and an anti-IAA monoclonal antibody, we confirmed that free IAA was present throughout style tissues, and distributed in the nucleus and cytoplasm of style cells. All these results suggested that hormones, especially IAA, play important roles in pollen tube growth of T. fournieri. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Detection of haplotypic variation and natural hybridization in halepensis-complex pine species using chloroplast simple sequence repeat (SSR) markers

In this investigation, nine chloroplast, paternally inherited simple-sequence repeat (cpSSR) markers were used to describe genetic variation of three closely related species belonging to the halepensis complex ( Pinus halepensis Ait., P. brutia Mill. and P. eldarica Medwed.). Both the infinite allele model (IAM) and stepwise-mutation model (SMM) have been applied to the analysis of the genetic structure of natural populations and the geographical distribution of haplotypic variation. SMM-based estimators performed better than IAM-based estimators for large values of within-population diversity and divergence between population pairs. Overall, large haplotypic variation and high genetic divergence were detected for both P. halepensis and P. brutia . The genetic structures of the three species are discussed with consideration to the evolutionary and ecological characteristics of these species. Three highly informative markers showing size variants distinguishing P. halepensis from the other two species were used to provide more information on the occurrence of natural hybridization in a Turkish sympatric population of P. halepensis and P. brutia . Strong evidence of introgression of ' halepensis ' chloroplast haplotypes into P. brutia seeds (but not vice versa) was detected. According to previous evidence from controlled crossings, matings between the above species seem to be successful only when P. halepensis is the pollen donor and P. brutia is the female parent (but not reciprocally). The existence of unidirectional gene flow in sympatric populations confirms previous evidence about partial reproductive barriers between P. halepensis and P. brutia . Implications of the above evidence for the evolutionary history of these species are discussed.     

A toxaphene-degrading bacterium related to Enterobacter cloacae, strain D1 isolated from aged contaminated soil in Nicaragua

Enterobacter sp. strain D1 is a facultative anaerobic, Gram-negative heterotrophic bacterium isolated from toxaphene-contaminated soil. This organism was identified and characterized through phylogenetic and taxonomic studies. Based on 16S rDNA analysis, the strain D1 was clustered closely with the species Enterobacter cloacae subsp. dissolvens (LMG 2683) and E. cloacae (ATCC 13047T). Strain D1 resembled these E. cloacae strains with respect to various biochemical and nutritional characteristics, but also exhibited differences. Moreover, strain D1 is able to grow and survive with toxaphene supplied in the medium in the range 3-96 mg/L. Amongst the chemical components of toxaphene, octachlorocamphenes, nonachlorobornanes and decachlorobornanes were seen to be rapidly metabolized, although levels of hexachlorocamphenes and heptachlorobornanes were found to be slowly degraded, and subsequently accumulated during the last stage of the cultivation.     

Effect of defoliation on grass growth. A quantitative review

This study describes the seasonal patterns of seed and cone abortion in natural Pinus halepensis trees and assesses the effects of pollen quantity, tree size and cone location on seed and cone abortion. The cone-set ratio was similar to the expected value, based on literature on woody perennials. The seed-ovule ratio, however, was almost twice as high as its expected value. The investment in protective cone elements was high and did not vary with the seed-ovule ratio of a cone. Pollination factors influenced cone abortion, as indicated by the high abortion rate at the end of the pollination period. Furthermore, abortive cones had lower pollination rates and lower pollen loads than well developed cones. Effects of resource availability were assessed as effects of tree size and cone position on twigs. Small trees aborted more cones than big trees and cone abortion was higher in apical cones than in basal cones. Abortion in P. halepensis is selective, mediated by both pollen quantity and resource availability. The relative importance of pollen and resources is suggested to be flexible, probably varying between trees and years. The high seed-ovule ratio of P. halepensis is the result of high pollination rate and selective abortion. The selective abortion might be due to the high allocation to protective cone elements relative to the allocation to seeds.     

Auxin abolishes inhibitory effects of methylcyclopropen and amino oxyacetic acid on pollen grain germination,pollen tube growth,and the synthesis of ACC in petunia

As established by us earlier, ethylene behaves as a regulator of germination, development, and growth of male gametophyte during the progamic phase of fertilization. However, the mechanisms of the regulation of these processes remain so far unstudied. It is believed that the main factor providing variety of the ethylene responses is its interaction with other phytohormones. According to our working hypothesis, ethylene controls germination of pollen grains (PGs) and growth of pollen tubes (PTs) by interacting with auxin, which, as the available data indicate, is likely a key regulator of plant cell polarization and morphogenesis and one of the factors modulating the biosynthesis of ethylene at the level of ACC-synthase gene expression. In the present work, on germinating in vitro male gametophyte and the pollen-stigma system for petunia (Petunia hybrida L.) effects of phytohormones (ethylene and IAA) and known blockers repressing ethylene reception (1-methylcyclopropene, 1-MCP), the synthesis of ACC (amino oxyacetic acid, AOA) and transport IAA (triyodbenzoynaya acid, TYBA) on PGs germination, PTs growth and the synthesis of ACC were investigated. According to the data obtained, exogenous ethylene and IAA stimulated both PGs germination and PTs growth. 1-MCP and TYBA completely inhibited the first process, whereas IAA abolished the inhibitory action of 1-MCP and AOA on both the above processes. Etrel only partially weakened the inhibitory effect of TYBA. Examination of ACC synthesis modulation with AOA showed that IAA does not affect the level of ACC in germinating in vitro male gametophyte and nonpollinated stigmas, while this phytohormone insignificantly raised the level of ACC and abolished the inhibitory effect of AOA on its synthesis in the pollenstigma system. Pollination of stigmas with the pollen preliminarily treated with 1-MCP led to 2.5-fold decline in both the rate of PT growth and the level of ACC. At the same time, IAA abolished the inhibitory action of 1-MCP recovering the synthesis of ACC and growth of PTs to the control values. All these results, taken together, provide evidence for the interaction of the signal transduction pathways of ethylene and auxin at the level of ACC biosynthesis in the course of germination and growth of petunia male gametophyte during the progamic phase of fertilization.     

利用大肠杆菌细胞工厂生产吲哚-3-乙酸的研究

目的:利用重组大肠杆菌全细胞转化色氨酸生产IAA.方法:在大肠杆菌胞内构建两条全新的IAA合成途径,即吲哚-3-乙酰胺(indole-3-acetamide,IAM)途径和色胺(tryptamine,TRP)途径.结果:IAM途径涉及两个酶,分别是色氨酸-2-单加氧酶(IAAM)和酰胺酶(AMI1),构建好的重组大肠杆...     

Phlebotomus (Adlerius) halepensis vector competence for Leishmania major and Le. tropica

In Eurasia, phlebotomine sandflies of the subgenus Adlerius (Diptera: Psychodidae) comprise about 20 known species. Some are suspected vectors of visceral leishmaniasis (VL) and at least one species has been implicated as a vector of cutaneous leishmaniasis (CL). We tested Phlebotomus (Adlerius) halepensis Theodor (Jordan strain) for CL vector competence, compared with three standard vectors: Phlebotomus (Phlebotomus) duboscqi N-L. from Senegal, Phlebotomus (Paraphlebotomus) sergenti Parrot from Turkey and the Neotropical Lutzomyia longipalpis (L. & N) (Jacobina strain). Sandfly females were membrane-fed on amastigote suspensions of Leishmania major Y. & S. and Le. tropica (Wright) (Kinetoplastida: Trypanosomatidae) and examined for parasite development 3, 6 and 10 days post-infection. Phlebotomus halepensis showed high susceptibility to both leishmanias, supporting typical suprapylarian parasite development similar to the other vectors. Phlebotomus halepensis infection rates were approximately 90% for Le. major and approximately 80% for Le. tropica, with high parasite densities. Development of infections was relatively fast, colonizing the thoracic midgut by 6 days post-bloodmeal in every case and reaching the stomodeal valve in >80% of flies. In late-stage infections, 10 days post-bloodmeal, nearly all P. halepensis females had cardia and stomodeal valve filled with very high numbers of parasites and some Le. tropica-infected females had promastigotes in the pharynx and proboscis. Host choice experiments in the laboratory showed that P. halepensis females fed readily on rat or rabbit and preferred the human forearm. In view of its vector competence and partial anthropophily, we infer that P. halepensis is a potential vector of cutaneous as well as visceral leishmaniases.     

Comparison of the Substrate Specificities of the β-Lactamases from Klebsiella aerogenes 1082E and Enterobacter cloacae P99

A potent beta-lactamase (EC 3.5.2.6) produced by a strain of Klebsiella aerogenes (K. pneumoniae), 1082E, isolated from a hospital patient, has been examined. Its properties were different from those of most gram-negative beta-lactamases previously reported. The enzyme has been partly purified, and its activity against a range of substrates has been compared with that of the enzyme from Enterobacter cloacae (Aerobacter cloacae) P99. The K. aerogenes enzyme, although predominantly a penicillinase, had a wide range of specificity. In addition to hydrolyzing the cephalosporins, it attacked the normally beta-lactamaseresistant compounds methicillin and cloxacillin as well as cephalosporin analogues with the same acyl substituents. The results obtained with the E. cloacae enzyme confirmed its cephalosporinase activity and showed that, unlike the enzyme from K. aerogenes, it was relatively inactive against the penicillins.     

UV-B 辐射对番茄花粉生活力的影响与内源激素和多胺的关系

以成熟期不同的两个番茄品种同辉(早熟型)和霞光(晚熟型)为试材, 模拟兰州地区12%和20%臭氧层减薄时增强的UVB辐射(分别为T1=2.54 KJ.m-2.d-1和T2=4.25 KJ.m-2.d-1), 研究了大田条件下增强UV-B辐射对其花粉生活力的影响以及雄蕊中4种内源激素(IAA、GAs、ZR和ABA)和多胺(Put、Spm和Spd)及脯氨酸的变化。结果表明: 辐射抑制了同辉的花粉萌发和花粉管伸长, 但只降低了霞光的花粉萌发率; 两种辐射明显降低了两品种番茄雄蕊中的GAs含量, 同时造成同辉雄蕊中Put和Spd含量明显增加, Spm和Put/Spd+Spm比值显著降低; 霞光中3种多胺含量都显著减少, 导致高辐射时Put/Spd+Spm比值上升; 同辉番茄雄蕊的脯氨酸含量不受影响, 但高辐射使霞光番茄雄蕊的脯氨酸含量降低。实验表明, 两品种番茄花粉生活力的变化与增强UV-B 辐射下雄蕊中GAs水平、Spm含量以及脯氨酸含量的减少有关。雄蕊中多胺和脯氨酸含量变化对UVB辐射的响应说明霞光品种对UV-B辐射更敏感。     

Role of heat tolerance and cone protection of seeds in the response of three pine species to wildfires

The post-fire regenerative ability of Pinus halepensis, Pinus nigra and Pinus sylvestris, three of the most important pine species present in the West Mediterranean basin, has been analyzed in the light of seed tolerance to different temperatures and times of exposure, and of seed position during the fire event (seeds inside cones versus free seeds). The combination of different fire intensities and degrees of seed protection allows us to draw different scenarios during the fire event: canopy scenarios (seeds inside cones), surface scenarios (seeds on the ground surface), and soil scenarios (seeds in the top soil layers). There were interspecific differences in the pattern of cone opening under the different heat treatments: cones of P. nigra and P. sylvestris showed similar percentages of opening, but considerably higher than those of P. halepensis. In the three species, seeds inside cones showed higher percentages of germination than those that were free, emphasizing the important role of cones in the protection of pine seeds from high temperatures. The percentage of germination decreased when both the temperature and the time of exposure increased, and there was also a significant species effect: P. halepensis showed higher germination rates than P. nigra, and both were higher than P. sylvestris. The overall scores of seed germination of these three pine species under the conditions tested suggests that their regeneration after fire should come either from the soil bank, or from the canopy bank, but rarely from the ground surface. As the existence of a permanent seed bank in Mediterranean pines is probably limited or nil, pine recruitment after fire appears to be mainly controlled by the existence of a canopy seed bank. The contribution of this canopy bank to the differences in postfire regeneration success of the three pine species is discussed in the light of their seeding phenology and the effects of fire severity on cone opening. The results obtained in this study contribute to explain the successful regeneration of P. halepensis, and the failure of P. nigra and P. sylvestris after fire.     

Extension of resistance to cefepime and cefpirome associated to a six amino acid deletion in the H-10 helix of the cephalosporinase of an Enterobacter cloacae clinical isolate

Enterobacter cloacae CHE, a clinical strain with overproduced cephalosporinase was found to be highly resistant to the new cephalosporins, cefepime and cefpirome (MICs> or =128 microg ml(-1)). The strain was isolated from a child previously treated with cefepime. The catalytic efficiency of the purified enzyme with the third-generation cephalosporins, cefepime and cefpirome, was 10 times higher than that with the E. cloacae P99 enzyme. This was mostly due to a decrease in K(m) for these beta-lactams. The clinical isolate produced large amounts of the cephalosporinase because introduction of the ampD gene decreased ampC expression and partially restored the wild-type phenotype. Indeed, MICs of cefepime and cefpirome remained 10 times higher than those for a stable derepressed clinical isolate (OUDhyp) transformed with an ampD gene. Sequencing of the ampC gene showed that 18 nucleotides had been deleted, corresponding to the six amino acids SKVALA (residues 289--294). According to the crystal structure of P99 beta-lactamase, this deletion was located in the H-10 helix. The ampR-ampC genes from the clinical isolates CHE and OUDhyp were cloned and expressed in Escherichia coli JM101. The MICs of cefpirome and cefepime of E. coli harboring ampC and ampR genes from CHE were 100--200 times higher than those of E. coli harboring ampC and ampR genes from OUDhyp. This suggests that the deletion, confirmed by sequencing of the ampC gene, is involved in resistance to cefepime and cefpirome. However, the high level of resistance to cefepime and cefpirome observed in the E. cloacae clinical isolate was due to a combination of hyperproduction of the AmpC beta-lactamase and structural modification of the enzyme. This is the first example of an AmpC variant conferring resistance to cefepime and cefpirome, isolated as a clinical strain.