Root Vascular Tissue Research Articles

Silicon modifies root anatomy, and uptake and subcellular distribution of cadmium in young maize plants.

Silicon (Si) has been shown to ameliorate the negative influence of cadmium (Cd) on plant growth and development. However, the mechanism of this phenomenon is not fully understood. Here we describe the effect of Si on growth, and uptake and subcellular distribution of Cd in maize plants in relation to the development of root tissues. Young maize plants (Zea mays) were cultivated for 10 d hydroponically with 5 or 50 µm Cd and/or 5 mm Si. Growth parameters and the concentrations of Cd and Si were determined in root and shoot by atomic absorption spectrometry or inductively coupled plasma mass spectroscopy. The development of apoplasmic barriers (Casparian bands and suberin lamellae) and vascular tissues in roots were analysed, and the influence of Si on apoplasmic and symplasmic distribution of (109)Cd applied at 34 nm was investigated between root and shoot. Si stimulated the growth of young maize plants exposed to Cd and influenced the development of Casparian bands and suberin lamellae as well as vascular tissues in root. Si did not affect the distribution of apoplasmic and symplasmic Cd in maize roots, but considerably decreased symplasmic and increased apoplasmic concentration of Cd in maize shoots. Differences in Cd uptake of roots and shoots are probably related to the development of apoplasmic barriers and maturation of vascular tissues in roots. Alleviation of Cd toxicity by Si might be attributed to enhanced binding of Cd to the apoplasmic fraction in maize shoots.

First Report of Verticillium Wilt Caused by Verticillium dahliae on Coleus verschaffeltii in Italy.

Coleus verschaffeltii Lem. (synonym C. blumei Benth., Plectranthus scutellaroides (L.) R. Br., and Solenostemon scutellarioides (L.) Codd), a perennial plant belonging to the Lamiaceae family, is used as a bedding plant for public gardens. The most popular cultivars produce speckled leaves of various colors. In October 2010, severe outbreaks of a previously unknown wilt were observed in a public garden at Torino (northern Italy) on 50 8-month-old plants. Plants were sprinkle irrigated. Initial symptoms were withering of leaves starting from the collar and brown streaks in the vascular tissue of roots, crown, and stem. Subsequently, infected tissues wilted and plants became stunted. Early leaf drop was observed and plants appeared bare, keeping few leaves only at the end of stems. Infected plants did not die but they lost the original ornamental aspect. Seventy percent of the plants were affected. Stems of 10 plants were disinfected with 1% sodium hypochlorite. Cross-sections through symptomatic vascular tissues were plated on potato dextrose agar amended with 25 ppm of streptomycin sulfate. After 10 days at 20 to 23°C, a fungus was consistently recovered from 90% of stems. Irregular, black microsclerotia, 29 to 76 × 14 to 52 (average 49 × 28) μm, developed in hyaline hyphae after 15 days of growth. Hyaline, elliptical, single-celled conidia, 3.9 to 7.2 × 1.7 to 2.8 (average 5.1 × 2.2) μm, developed on verticillate conidiophores with three phialides at each node. On the basis of these morphological characteristics, the fungus was identified as Verticillium dahliae (3). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 (4) and sequenced. BLASTn analysis (1) of the 491-bp segment showed a 99% homology with the sequence of V. dahliae (Accession No. GU461634). The ITS nucleotide sequence of our isolate has been assigned the GenBank Accession No. JF704205. Pathogenicity tests were performed twice using 45-day-old plants obtained from seeds of C. verschaffeltii grown in 1-liter pots containing a 50:20:20:10 steamed mix of peat moss/pumice/pine bark/clay. Roots of 10 healthy plants were immersed in a conidial suspension (1.7 × 107 ml-1) of one culture of V. dahliae isolated from infected plants. Ten plants immersed in sterile water served as controls. Plants were maintained in a glasshouse at daily average temperatures between 20 and 28°C and relative humidity between 50 and 80%. First wilt symptoms and vascular discoloration in the roots, crown, and stems developed 20 days after inoculation. V. dahliae was consistently reisolated from infected vascular tissues of crown and stems of symptomatic plants. Noninoculated plants remained healthy. To our knowledge, this is the first report of Verticillium wilt on C. verschaffeltii in Italy. Verticillium wilt had been previously reported on S. scutellaroides in the United States (2). At this time, the economic importance of Verticillium wilt on C. verschaffeltii in Italy is limited.

Dieback and mortality of South African fynbos shrubs is likely driven by a novel pathogen and pathogen‐induced hydraulic failure

AbstractWe examined whether extensive dry season dieback and mortality in a South African fynbos community were due to drought or pathogen attack. Plant dieback and mortality have been reported elsewhere in similar plant communities suggesting potential for a widespread climatic or biotic threat to this community. We collected tissue samples from Brunia noduliflora, the dominant plant in the community, and cultured them for potential plant pathogens. We also measured dry season predawn and midday water potentials of healthy and stressed plants and constructed pressure‐volume curves to assess turgor loss point. Plant stress and mortality were monitored over a 2‐year study period. Both healthy plants and plants that displayed moderate signs of stress had dry season predawn water potentials well above their turgor loss point suggesting plants were not water stressed. However, plants displaying >60% crown dieback had much lower water potentials (as low as −12 MPa). A previously undescribed fungus (Pythium sp.) was isolated from the root vascular tissue of all stressed plants and was not present in healthy plants. The proximate cause of plant stress was likely pathogen‐induced, while the ultimate cause of plant death appears to be extreme water stress. The present study suggests that Brunia (Bruniceae), Leucadendron (Proteaceae) and Erica (Ericaceae), all emblematic and dominant genera within the diverse fynbos community, may be susceptible to Pythium infection. This may pose a serious threat to communities already threatened by climate change.

Anatomical Response of St. Augustinegrass to Aminocyclopyrachlor Treatment

Aminocyclopyrachlor (AMCP) is a synthetic auxin herbicide that controls primarily broadleaf (eudicotyledonous) weeds. Previous research indicates that St. Augustinegrass is unacceptably injured by AMCP. In light of the fact that synthetic auxin herbicides usually are safe when applied to monocotyledons, the mechanism for this injury is not fully understood. Anatomical response of St. Augustinegrass to AMCP was investigated using light microscopy. Apical meristem node tissue responded with callus tissue proliferation, abnormal location and development of the apical meristem, necrosis of the developing vascular tissue, vascular parenchyma proliferation, and xylem gum blockages. Node tissues away from the apical meristem responded with xylem gum blockages and the stimulation of lateral meristems and adventitious root formation. Root tip response to AMCP treatment was characterized by a loss of organization. Root tip apical meristem and vascular tissue maturation was disorganized. Additionally, lateral root generation occurred abnormally close to the root tip. These responses impair affected tissue functionality. Mature tissue was unaffected by AMCP treatment. All of these responses are characteristic of synthetic auxin herbicide treatment to other susceptible species. This research indicates that AMCP treatment results in St. Augustinegrass injury and subsequent death through deleterious growth stimulation and concomitant vascular inhibition.

Acid phosphatases and growth of barley (Hordeum vulgare L.) cultivars under diverse phosphorus nutrition

The morphological and physiological responses of barley to moderate Pi deficiency and the ability of barley to grow on phytate were investigated. Barley cultivars (Hordeum vulgare L., Promyk, Skald and Stratus) were grown for 1–3 weeks on different nutrient media with contrasting phosphorus source: KH2PO4 (control), phytic acid (PA) and without phosphate (−P). The growth on −P medium strongly decreased Pi concentration in the tissues; culture on PA medium generally had no effect on Pi level. Decreased content of Pi reduced shoot and root mass but root elongation was not affected; Pi deficit had slightly greater impact on growth of barley cv. Promyk than other varieties. Barley varieties cultured on PA medium showed similar growth to control. Extracellular acid phosphatase activities (APases) in −P roots were similar to control, but in PA plants were lower. Histochemical visualization indicated for high APases activity mainly in the vascular tissues of roots and in rhizodermis. Pi deficiency increased internal APase activities mainly in shoot of barley cv. Stratus and roots of cv Promyk; growth on PA medium had no effect or decreased APase activity. Protein extracts from roots and shoots were run on native discontinuous PAGE to determine which isoforms may be affected by Pi deficiency or growth on PA medium; two of four isoforms in roots were strongly induced by conditions of Pi deficit, especially in barley cv. Promyk. In conclusion, barley cultivars grew equally well both on medium with Pi and where the Pi was replaced with phytate and only slightly differed in terms of acclimation to moderate deficiency of phosphate; they generally used similar pools of acid phosphatases to acquire Pi from external or internal sources.

Efficient chimeric promoters derived from full-length and sub-genomic transcript promoters of Figwort mosaic virus (FMV)

Addition of multiple repeats of the FS3 upstream activation sequence (FS3-UAS, −270 to −60) intra-molecularly to the TATA containing core-domain of the FS3 (−151 to +31) promoter resulted in 2–3-folds enhanced promoter activity. The chimeric promoter, FS3-UAS-3X with maximum activity, showed 3.31 times stronger activity in root vascular tissue compared to FS3 promoter and could be used efficiently in translational research.

Study on Mechanisms of Colonization of Nitrogen-Fixing PGPB, Klebsiella pneumoniae NG14 on the Root Surface of Rice and the Formation of Biofilm

Plant growth-promoting bacteria (PGPB) refer to the bacteria beneficial to plants, and they may affect the growth and development of plants directly or indirectly. This article studied the activities of nitrogen fixation and colonization of a strain of PGPB, Klebsiella pneumoniae NG14, which was isolated from the rice root surface. The results showed that NG14 harbouring the nifH gene had nitrogenase activity, (15)N(2)-fixing activity, and was able to colonize on the root surface and within the cavity of root vascular tissues of rice. Using proteomics technology to study the differences and changes of membrane proteins (MP) of NG14 bacterial biofilm in non-biological surface, 28 proteins showing significant differences before and after the formation of bacterial biofilm have been identified, in which the precursors of membrane pore protein OmpC relevant to osmotic stress resistance was up-regulated. This study would have positive significance on further understanding of the direct and indirect promotion effects of PGPB and related mechanisms.

Induced thyme product prevents VEGF-induced migration in human umbilical vein endothelial cells

Compounds with anti-angiogenic properties are useful in combating cancer by preventing new blood vessel formation to support the tumor. In this report we introduce a rapid method for screening potential anti-angiogenic compounds in a model system that stimulates the production of secondary defense chemicals in plants. This methodology identified an inducible vascular factor (IVF3), which was found to be inhibitory in all of the model systems tested. Thyme plants were exposed to highly vascular mint plants and the methanol extracts were analyzed by reverse phase HPLC. The thyme compounds induced by the invading mint tissue, and not present in the thyme plants grown alone, were tested in a vertical plate assay measuring root length as a quantitative assay for drug sensitivity. The HPLC-purified extract, referred to as IVF3, reduced the growth of root vascular tissue compared to the control and vehicle control, and 50% as well as known angiogenesis inhibitors, VEGF receptor tyrosine kinase inhibitor and amiloride hydrochloride. Extracted compounds that were effective inhibitors of plant roots were assayed in Madin Darby canine kidney epithelial cells (MDCK) for toxicity, and in human umbilical vein endothelial cells (HUVEC) for their effect on migration. IVF3 was effective at limiting HUVEC migration in VEGF-stimulated cultures. In vivo video capture of intersegmental vessel circulation between 48 and 72 h post fertilization in the developing vasculature of zebrafish embryos showed IVF3 also significantly reduced ISV functional circulation. This report demonstrates the anti-angiogenic effects of IVF3 extract in endothelial cells and in an intact vertebrate model for angiogenesis.

Functional analysis of Gossypium hirsutum cellulose synthase catalytic subunit 4 promoter in transgenic Arabidopsis and cotton tissues

Gossypium hirsutum cellulose synthase catalytic subunit 4 ( GhCesA4) plays an important role in cellulose biosynthesis during cotton fiber development. The transcript levels of GhCesA4 are significantly up-regulated as secondary cell wall cellulose is produced in developing cotton fibers. To understand the molecular mechanisms involved in transcriptional regulation of GhCesA4, β-glucuronidase (GUS) activity regulated by a GhCesA4 promoter (−2574/+56) or progressively deleted promoters were determined in both cotton tissues and transgenic Arabidopsis. The spatial regulation of GhCesA4 expression was similar between cotton tissues and transgenic Arabidopsis. GUS activity regulated by the GhCesA4 promoter (−2574/+56) was found in trichomes and root vascular tissues in both cotton and transgenic Arabidopsis. The −2574/−1824 region was responsible for up-regulation of GhCesA4 expression in trichomes and root vascular tissues in transgenic Arabidopsis. The −1824/−1355 region negatively regulated GhCesA4 expression in most Arabidopsis vascular tissues. For vascular expression in stems and leaves, the −898/−693 region was required. The −693/−320 region of the GhCesA4 promoter was necessary for basal expression of GhCesA4 in cotton roots as well as Arabidopsis roots. Exogenous phytohormonal treatments on transgenic Arabidopsis revealed that phytohormones may be involved in the differential regulation of GhCesA4 during cotton fiber development.

Bidirectional cell signaling and gene regulation in the root vascular tissue patterning and growth

Bidirectional cell signaling and gene regulation in the root vascular tissue patterning and growth

Tissue expression analysis of FeMT3, a drought and oxidative stress related metallothionein gene from buckwheat ( Fagopyrum esculentum)

Metallothionein type 3 (MT3) expression has previously been detected in leaves, fruits, and developing somatic embryos in different plant species. However, specific tissular and cellular localization of MT3 transcripts have remained unidentified. In this study, in situ RNA–RNA analysis revealed buckwheat metallothionein type 3 ( FeMT3) transcript localization in vascular elements, mesophyll and guard cells of leaves, vascular tissue of roots and throughout the whole embryo. Changes in FeMT3 mRNA levels in response to drought and oxidative stress, as well as ROS scavenging abilities of the FeMT3 protein in yeast were also detected, indicating possible involvement of FeMT3 in stress defense and ROS related cellular processes.

Expression analysis and functional characterization of the monosaccharide transporters, OsTMTs, involving vacuolar sugar transport in rice (Oryza sativa)

In Arabidopsis, the compartmentation of sugars into vacuoles is known to be facilitated by sugar transporters. However, vacuolar sugar transporters have not been studied in detail in other plant species. To characterize the rice (Oryza sativa) tonoplast monosaccharide transporters, OsTMT1 and OsTMT2, we analysed their subcellular localization using green fluorescent protein (GFP) and expression patterns using reverse-transcription polymerase chain reaction (RT-PCR), performed histochemical beta-glucuronidase (GUS) assay and in situ hybridization analysis, and assessed sugar transport ability using isolated vacuoles. Expression of OsTMT-GFP fusion protein in rice and Arabidopsis revealed that the OsTMTs localize at the tonoplast. Analyses of OsTMT promoter-GUS transgenic rice indicated that OsTMT1 and OsTMT2 are highly expressed in bundle sheath cells, and in vascular parenchyma and companion cells in leaves, respectively. Both genes were found to be preferentially expressed in the vascular tissues of roots, the palea/lemma of spikelets, and in the main vascular tissues and nucellar projections on the dorsal side of the seed coats. Glucose uptake studies using vacuoles isolated from transgenic mutant Arabidopsis (tmt1-2-3) expressing OsTMT1 demonstrated that OsTMTs are capable of transporting glucose into vacuoles. Based on expression analysis and functional characterization, our present findings suggest that the OsTMTs play a role in vacuolar glucose storage in rice.

First Report of Verticillium Wilt Caused by Verticillium dahliae on Figmarigold in Italy.

Lampranthus spp. N.B. Brown (figmarigold) belongs to the Aizoaceae family and is grown as a ground cover in gardens or as a potted plant. In January 2009, severe outbreaks of a previously unknown wilt were observed at a commercial farm in Liguria (northern Italy) where 7-month-old potted plants were grown outdoors in a mix of peat/clay/pumice at pH 6.5. In cultivars with pink flowers, 12% of plants were affected, while only a few cultivars with red flowers were diseased. Initial symptoms were yellowing of external leaves and brown or black streaks in the vascular tissue of roots, crown, and leaves. Subsequently, infected tissues wilted and stopped growing, stems and leaves appeared desiccated, and infected plants died. Stems of 10 pink-flowered plants were severed with a knife, cut ends sealed with wax, and surfaces disinfected with 1% sodium hypochlorite. Cross-sections (1 mm long) through symptomatic vascular tissue were plated onto potato dextrose agar. After 10 days at 22°C, 90% of the stems tested positive for Verticillium. Irregular, dark microsclerotia, 22 to 128 × 13 to 66 (average 51 × 29) μm, developed in hyaline hyphae after 10 days of growth at 22 ± 1°C (12-h photoperiod). Hyaline, elliptical, single-celled conidia, 2.9 to 4.8 × 1.3 to 2.4 (average 4.1 × 1.7) μm, developed on verticillate conidiophores. On the basis of these morphological characteristics, the fungus was identified as V. dahliae (3). The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4/ITS6 (2) and sequenced. BLASTn analysis (1) of the 476-bp segment showed a 100% homology with the sequence of V. dahliae. The nucleotide sequence has been assigned GenBank Accession No. GQ 149479. Pathogenicity tests were performed twice using five 40-day-old plants of a pink-flower cultivar of a Lampranthus sp. grown in 1-liter pots containing a 50:20:20:10 mix of peat moss/pumice/pine bark cortex/clay. The substrate was infested with a conidial suspension (1.0 × 107/ml) of one isolate of V. dahliae recovered from infected plants. Inoculum (50 ml) were added to each pot, drenching the top of the soil. Noninoculated plants served as controls. Plants (five per treatment) were maintained in a glasshouse at daily average temperatures between 20 and 26°C and at 50 to 70% relative humidity. The first wilt symptoms and a vascular discoloration in the roots, crown, and veins developed 30 days after inoculation. V. dahliae was consistently reisolated. Noninoculated plants remained healthy. In a second test, the susceptibility of purple-, white-, yellow-, red-, and orange-flowered cultivars was tested. Ten rooted cuttings of each cultivar were inoculated as described above. The severity of Verticillium wilt was evaluated and each cultivar was classified as resistant, partially resistant, average susceptible, susceptible, or highly susceptible. All tested cultivars were susceptible or highly susceptible to Verticillium. Only the purple cultivar showed an average susceptibility. To our knowledge, this is the first report of Verticillium wilt on Lampranthus spp. in Italy as well as worldwide. Today, the economic importance of Verticillium wilt on figmarigold in Italy is still limited.

Uptake, localisation and physiological changes in response to copper excess in Erica andevalensis

Copper uptake, localisation and biochemical and physiological traits were studied in hydroponically-grown Erica andevalensis plants at different increasing concentrations of Cu (1 µM, 50 µM, 100 µM, 250 µM, and 500 µM). Increasing Cu concentration in the nutrient medium led to a significative reduction in plant growth rate, an increase in root Cu concentration, leaf photosynthetic pigments and root peroxidase activity. Copper accumulation followed the pattern roots>stems>leaves, a typical behaviour of metal-excluders. Copper treatments led to significant changes in the free amino acid composition in shoots and roots and the concentration of polyamines in shoots. Analysis by scanning electron microscopy coupled with elemental X-ray analysis (SEM–EDX) showed a partial restriction of upward Cu transport by root vascular tissues. In leaf tissues, Cu mostly accumulated in the abaxial epidermis, suggesting a mechanism of compartmentalization to restrict mesophyll accumulation. The toxic effects of excess Cu were avoided to a certain extent by root immobilization, tissue compartmentalization, synthesis of complexing amino acids and induction of enzymes to prevent oxidative damage are among mechanisms adopted by Erica andevalensis to thrive in acidic-metalliferous soils.

Induction of Lead‐Binding Phytochelatins in Vetiver Grass [Vetiveria zizanioides (L.)

Elevated lead (Pb) concentrations in residential houseyards around house walls painted with Pb-based pigments pose serious human health risks, especially to children. Vetiver grass (Vetiveria zizanioides L.) has shown promise for use in in situ Pb phytoremediation efforts. However, little is known about the biochemical mechanisms responsible for the observed high Pb tolerance by vetiver. We hypothesized that vetiver exposure to Pb induced the synthesis of phytochelatins (PC(n)) and the formation of Pb-PC(n) complexes, alleviating the phytotoxic effects of free Pb ions. Our main objective was to identify PC(n) and Pb-PC(n) complexes in root and shoot compartments of vetiver grass using high-performance liquid chromatography coupled to electrospray mass spectrometry (HPLC-ES-MS). After 7 d of exposure to Pb, vetiver accumulated up to 3000 mg Pb kg(-1) in shoot tissues, but much higher Pb concentrations were measured in root ( approximately 20,000 mg kg(-1)), without phytotoxic symptoms. Scanning electron micrographs showed Pb deposition in the vascular tissues of root and shoot, suggesting Pb translocation to shoot. Collision-induced dissociation analyses in MS/ MS mode during HPLC-ES-MS analysis allowed for the confirmation of four unique PC(n) (n = 1-4) based on their respective amino acid sequence. The high tolerance of vetiver grass to Pb was attributed to the formation of PC(n) and Pb-PC(n) complexes within the plant tissues, using ES-MS and Pb mass isotopic patterns. These data illustrate the mechanism of high Pb tolerance by vetiver grass, suggesting its potential usefulness for the remediation of Pb-contaminated residential sites.

Abstract A55: A screening method for identifying potential angiogenesis inhibitors

Abstract A55 Objective Plants produce chemical compounds to prevent encroachment by competitors. These turf wars that generate defense products in plants have potential medicinal value for humans. For example, compounds with anti-angiogenic properties are useful in combating cancer by preventing new blood vessel formation to support the tumor. The search for new angiogenesis inhibitors is ongoing, and utilizes a variety of time-intensive cell culture models. In this report we introduce a rapid, inexpensive method for screening potential anti-angiogenic compounds in a model system that stimulates the production of secondary defense chemicals in plants, thus increasing the potential yield of effective compounds. Plant chemicals under investigation for anti-angiogenic activity include flavonoids, a broad group of compounds that are present in most photosynthesizing cells of plants. Flavonoids share a common phenyl-chromanone structure (C6-C3-C6), and include thousands of compounds, subdivided into 6 major categories; flavones, flavonols, isoflavones, flavanols, flavanones, and anthocyanins. Humans do not synthesize flavonoids, but derive an average of 1 gram per day in their diet. Their reported pharmacological properties include antioxidative, anti-inflammatory and antiproliferative properties. Methods In order to induce defense chemical production, we chose thyme and mint plants to combine because thyme was observed to inhibit the spread of mint, a highly invasive vascular plant. The thyme plants grown alone, as well as the mint alone, were analyzed as controls. The plant leaves and stems were extracted and the flavonoid products were analyzed by reverse phase HPLC. The flavonoids that appear in the thyme in the presence of mint, that are not present in the thyme plants grown alone, were considered induced by the presence of mint. The induced compounds were collected and tested in a vertical plate assay that measures root length as a quantitative assay for drug sensitivity. The compounds were applied to the vascular root tissue of mint seedlings 5-7 days after germination. Results Of eight Thymus vulgaris compounds that were induced by the presence of mint, 5 were tested by bioassay, and one was found to have an inhibitory effect. Known angiogenesis inhibitors, VEGF receptor tyrosine kinase and amelioride hydrochloride, significantly reduced root growth compared to the control. An HPLC-isolated compound b, which eluted at 15.2 minutes, reduced the growth of root vascular tissue compared to the control and vehicle control, and 50% as well as known angiogenesis inhibitors. Conclusion This report demonstrates that induced secondary defense chemicals produced by thyme plants include flavonoid compounds. Useful inhibitors may be identified with this rapid, sensitive, and inexpensive technique. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A55.

First Report of Verticillium Wilt Caused by Verticillium dahliae on Rudbeckia fulgida (Orange Coneflower) in Italy.

Rudbeckia fulgida (common name orange coneflower) is an herbaceous perennial (Asteraceae) grown in full sun in perennial borders in gardens. At the end of the summer of 2007, in a public garden located in Turin (northern Italy), symptoms of vascular wilt and stunting were observed on approximately 80% of the plants grown in a mixed border. Initial symptoms were yellowing of external leaves and brown or black streaks in the vascular tissue of roots, crown, and leaves. A fungus was consistently and readily isolated on potato dextrose agar from symptomatic vascular tissue previously disinfested in 1% sodium hypochloride. Ovoid, dark microsclerotia, 41 to 108 μm, developed in hyaline hyphae after 10 days of growth at 23°C (12 h of light and 12 h of dark). Hyaline, elliptical, single-celled conidia, 3.2 to 7.3 × 2.1 to 3.7 (average 4.7 × 2.8) μm, developed on verticillate conidiophores. On the basis of these morphological characteristics, the fungus was identified as Verticillium dahliae (4). The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4/ITS6 and sequenced. BLASTn analysis (1) of the 530 bp showed a 100% homology with the sequence of V. dahliae. The nucleotide sequence has been assigned GenBank Accession No. EU 627007. Healthy, 30-day-old R. fulgida plants were grown in a steam-disinfested mix of sphagnum peat:pomix:pine bark:clay (50:20:20:10) infested with a conidial suspension (1.5 × 106/ml) of three isolates of V. dahliae isolated from infected plants. Noninoculated plants served as controls. Plants (16 per treatment) were grown in pots (3 liter vol) and maintained in a glasshouse at temperatures between 22 and 25°C and relative humidity between 50 and 70%. First wilt symptoms and vascular discoloration in the roots, crown, and veins developed 17 days after inoculation. Noninoculated plants remained healthy. The pathogenicity tests were carried out twice. To our knowledge, this is the first report in Italy of Verticillium wilt on R. fulgida. Wilts caused by V. dahliae on R. laciniata in Poland (3) and V. albo-atrum on R. hirta in the United States (2) were previously reported. The importance and economic impact of this disease is currently limited but may increase because of the popularity of Rudbeckia spp. in private and public parks.

Characteristic and Expression Analysis of a Metallothionein Gene, OsMT2b, Down-Regulated by Cytokinin Suggests Functions in Root Development and Seed Embryo Germination of Rice

Metallothioneins (MTs) are low molecular mass and cysteine-rich metal-binding proteins known to be mainly involved in maintaining metal homeostasis and stress responses. But, their functions in higher plant development are scarcely studied. Here, we characterized rice (Oryza sativa) METALLOTHIONEIN2b (OsMT2b) molecularly and found that its expression was down-regulated by cytokinins. OsMT2b was preferentially expressed in rice immature panicles, scutellum of germinating embryos, and primordium of lateral roots. In contrast with wild-type plants, OsMT2b-RNA interference (RNAi) transgenic plants had serious handicap in plant growth and root formation, whereas OsMT2b-overexpressing transformants were dwarfed and presented more adventitious roots and big lateral roots. The increased cytokinin levels in RNAi plants and decreased cytokinin levels in overexpressing plants were confirmed by high-performance liquid chromatography quantitative analysis in the roots of wild-type and transgenic plants. In RNAi plants, localization of isopentenyladenosine, a kind of endogenous cytokinin, in roots and germinating embryos expanded to the whole tissues, whereas in overexpressing plants, the isopentenyladenosine signals were very faint in the vascular tissues of roots and scutellum cells of germinating embryos. In vitro culture of embryos could largely resume the reduced germination frequency in RNAi plants but had no obvious change in overexpressing plants. Taken together, these results indicate a possible feedback regulation mechanism of OsMT2b to the level of endogenous cytokinins that is involved in root development and seed embryo germination of rice.

ECA3, a Golgi-Localized P2A-Type ATPase, Plays a Crucial Role in Manganese Nutrition in Arabidopsis

Calcium (Ca) and manganese (Mn) are essential nutrients required for normal plant growth and development, and transport processes play a key role in regulating their cellular levels. Arabidopsis (Arabidopsis thaliana) contains four P(2A)-type ATPase genes, AtECA1 to AtECA4, which are expressed in all major organs of Arabidopsis. To elucidate the physiological role of AtECA2 and AtECA3 in Arabidopsis, several independent T-DNA insertion mutant alleles were isolated. When grown on medium lacking Mn, eca3 mutants, but not eca2 mutants, displayed a striking difference from wild-type plants. After approximately 8 to 9 d on this medium, eca3 mutants became chlorotic, and root and shoot growth were strongly inhibited compared to wild-type plants. These severe deficiency symptoms were suppressed by low levels of Mn, indicating a crucial role for ECA3 in Mn nutrition in Arabidopsis. eca3 mutants were also more sensitive than wild-type plants and eca2 mutants on medium lacking Ca; however, the differences were not so striking because in this case all plants were severely affected. ECA3 partially restored the growth defect on high Mn of the yeast (Saccharomyces cerevisiae) pmr1 mutant, which is defective in a Golgi Ca/Mn pump (PMR1), and the yeast K616 mutant (Deltapmc1 Deltapmr1 Deltacnb1), defective in Golgi and vacuolar Ca/Mn pumps. ECA3 also rescued the growth defect of K616 on low Ca. Promoter:beta-glucuronidase studies show that ECA3 is expressed in a range of tissues and cells, including primary root tips, root vascular tissue, hydathodes, and guard cells. When transiently expressed in Nicotiana tabacum, an ECA3-yellow fluorescent protein fusion protein showed overlapping expression with the Golgi protein GONST1. We propose that ECA3 is important for Mn and Ca homeostasis, possibly functioning in the transport of these ions into the Golgi. ECA3 is the first P-type ATPase to be identified in plants that is required under Mn-deficient conditions.

Root and Stem Infection of Rhododendron from Potting Medium Infested with Phytophthora ramorum.

Phytophthora ramorum has been detected in soil and potting media, but the potential for root infections is not fully understood. To determine whether the root system could become infected and transmit disease, rhododendron 'Nova Zembla' plants grown from rooted cuttings and native Pacific rhododendron (Rhododendron macrophyllum) plants grown from seed were transplanted into a potting medium artificially infested with P. ramorum. Inoculum consisted of V8-brothvermiculite cultures of P. ramorum, chopped infected leaves, or zoospores. Plants were watered from the bottom to prevent splash dispersal of inoculum onto stems and foliage. Both infested amendments and applications of zoospores resulted in plant mortality within 3 to 7 weeks. P. ramorum was isolated from hair roots, large roots, and stems above and below the potting medium surface. Noninoculated control plants remained healthy and did not yield P. ramorum. Epifluorescence microscopy of tissue culture plantlets inoculated in vitro revealed attraction of zoospores to wounds and root primordia, and colonization of the cortex and vascular tissues of roots and stems, including the xylem. Transmission of P. ramorum from infested potting media to stems via infected, symptomless root tissue demonstrates the need to monitor potting media for presence of the pathogen to prevent spread of P. ramorum on nursery stock.