Cruciferous Weeds Do Not Act as Major Reservoirs of Inoculum for Black Rot Outbreaks in New York State.

Cruciferous weeds have been shown to harbor diverse Xanthomonas campestris pathovars, including the agronomically damaging black rot of cabbage pathogen, X. campestris pv. campestris. However, the importance of weeds as inoculum sources for X. campestris pv. campestris outbreaks in New York remains unknown. To determine if cruciferous weeds act as primary reservoirs for X. campestris pv. campestris, fields that were rotating between cabbage or had severe black rot outbreaks were chosen for evaluation. Over a consecutive 3-year period, 148 cruciferous and noncruciferous weed samples were collected at 34 unique sites located across five New York counties. Of the 148 weed samples analyzed, 48 X. campestris isolates were identified, with a subset characterized using multilocus sequence analysis. All X. campestris isolates originated from weeds belonging to the Brassicaceae family, with predominant weed hosts being shepherd's purse (Capsella bursa-pastoris), wild mustard (Sinapis arvensis), yellow rocket (Barbarea vulgaris), and pennycress (Thlaspi arvense). Identifying pathogenic X. campestris weed isolates was rare, with only eight isolates causing brown necrotic leaf spots or typical V-shaped lesions on cabbage. There was no evidence of cabbage-infecting weed isolates persisting in an infected field by overwintering in weed hosts; however, similar cabbage and weed X. campestris haplotypes were identified in the same field during an active black rot outbreak. X. campestris weed isolates are genetically diverse both within and between fields, but our findings indicate that X. campestris weed isolates do not appear to act as primary sources of inoculum for B. oleracea fields in New York.

Culturable endophytic bacteria enhance Ni translocation in the hyperaccumulator Noccaea caerulescens.

In this work, both culture-dependent and independent approaches were used to identify and isolate endophytic bacteria from roots of the Ni hyperaccumulator Noccaea caerulescens. A total of 17 isolates were cultured from root samples, selected for tolerance to 6mM Ni and grouped by restriction analysis of 16S rDNA. Bacterial species cultivated from roots belonged to seven genera, Microbacterium, Arthrobacter, Agreia, Bacillus, Sthenotrophomonas, Kocuria and Variovorax. The culture-independent approach confirmed the presence of Microbacterium and Arthrobacter while only other five clones corresponding to different amplified ribosomal DNA restriction patterns were detected. Five selected highly Ni-resistant bacteria showing also plant growth promoting activities, were inoculated into seeds of N. caerulescens, and in vivo microscopic analysis showed rapid root colonisation. Inoculated plants showed increased shoot biomass, root length and root-to-shoot Ni translocation. Root colonisation was also evident, but not effective, in the non-hyperaccumulating Thlaspi perfoliatum. Seed inoculation with selected Ni-resistant endophytic bacteria may represent a powerful tool in phytotechnologies, although transferring it to biomass species still requires further studies and screening.

Uncoupling of reactive oxygen species accumulation and defence signalling in the metal hyperaccumulator plant Noccaea caerulescens.

The metal hyperaccumulator plant Noccaea caerulescens is protected from disease by the accumulation of high concentrations of metals in its aerial tissues, which are toxic to many pathogens. As these metals can lead to the production of damaging reactive oxygen species (ROS), metal hyperaccumulator plants have developed highly effective ROS tolerance mechanisms, which might quench ROS-based signals. We therefore investigated whether metal accumulation alters defence signalling via ROS in this plant. We studied the effect of zinc (Zn) accumulation by N. caerulescens on pathogen-induced ROS production, salicylic acid accumulation and downstream defence responses, such as callose deposition and pathogenesis-related (PR) gene expression, to the bacterial pathogen Pseudomonas syringae pv. maculicola. The accumulation of Zn caused increased superoxide production in N. caerulescens, but inoculation with P. syringae did not elicit the defensive oxidative burst typical of most plants. Defences dependent on signalling through ROS (callose and PR gene expression) were also modified or absent in N. caerulescens, whereas salicylic acid production in response to infection was retained. These observations suggest that metal hyperaccumulation is incompatible with defence signalling through ROS and that, as metal hyperaccumulation became effective as a form of elemental defence, normal defence responses became progressively uncoupled from ROS signalling in N. caerulescens.

Endogenous jasmonic and salicylic acids levels in the Cd-hyperaccumulator Noccaea (Thlaspi) praecox exposed to fungal infection and/or mechanical stress.

KEY MESSAGE: Sensitivity to Erysiphe in Noccaea praecox with low metal supply is related to the failure in enhancing SA. Cadmium protects against fungal-infection by direct toxicity and/or enhanced fungal-induced JA signaling. Metal-based defense against biotic stress is an attractive hypothesis on evolutionary advantages of plant metal hyperaccumulation. Metals may compensate for a defect in biotic stress signaling in hyperaccumulators (metal-therapy) by either or both direct toxicity to pathogens and by metal-induced alternative signaling pathways. Jasmonic acid (JA) and salicylic acid (SA) are well-established components of stress signaling pathways. However, few studies evaluate the influence of metals on endogenous concentrations of these defense-related hormones. Even less data are available for metal hyperaccumulators. To further test the metal-therapy hypothesis we analyzed endogenous SA and JA concentrations in Noccaea praecox, a cadmium (Cd) hyperaccumulator. Plants treated or not with Cd, were exposed to mechanical wounding, expected to enhance JA signaling, and/or to infection by biotrophic fungus Erysiphe cruciferarum for triggering SA. JA and SA were analyzed in leaf extracts using LC-ESI(-)-MS/MS. Plants without Cd were more susceptible to fungal attack than plants receiving Cd. Cadmium alone tended to increase leaf SA but not JA. Either or both fungal attack and mechanical wounding decreased SA levels and enhanced JA in the Cd-rich leaves of plants exposed to Cd. High leaf Cd in N. praecox seems to hamper biotic-stress-induced SA, while triggering JA signaling in response to fungal attack and wounding. To the best of our knowledge, this is the first report on the endogenous JA and SA levels in a Cd-hyperaccumulator exposed to different biotic and abiotic stresses. Our results support the view of a defect in SA stress signaling in Cd hyperaccumulating N. praecox.

Metal hyperaccumulation armors plants against disease.

Metal hyperaccumulation, in which plants store exceptional concentrations of metals in their shoots, is an unusual trait whose evolutionary and ecological significance has prompted extensive debate. Hyperaccumulator plants are usually found on metalliferous soils, and it has been proposed that hyperaccumulation provides a defense against herbivores and pathogens, an idea termed the 'elemental defense' hypothesis. We have investigated this hypothesis using the crucifer Thlaspi caerulescens, a hyperaccumulator of zinc, nickel, and cadmium, and the bacterial pathogen Pseudomonas syringae pv. maculicola (Psm). Using leaf inoculation assays, we have shown that hyperaccumulation of any of the three metals inhibits growth of Psm in planta. Metal concentrations in the bulk leaf and in the apoplast, through which the pathogen invades the leaf, were shown to be sufficient to account for the defensive effect by comparison with in vitro dose-response curves. Further, mutants of Psm with increased and decreased zinc tolerance created by transposon insertion had either enhanced or reduced ability, respectively, to grow in high-zinc plants, indicating that the metal affects the pathogen directly. Finally, we have shown that bacteria naturally colonizing T. caerulescens leaves at the site of a former lead-zinc mine have high zinc tolerance compared with bacteria isolated from non-accumulating plants, suggesting local adaptation to high metal. These results demonstrate that the disease resistance observed in metal-exposed T. caerulescens can be attributed to a direct effect of metal hyperaccumulation, which may thus be functionally analogous to the resistance conferred by antimicrobial metabolites in non-accumulating plants.

Changes in elemental uptake and arbuscular mycorrhizal colonisation during the life cycle of Thlaspi praecox Wulfen.

Elemental uptake and arbuscular mycorrhizal (AM) colonisation were studied during the life cycle of field collected Cd/Zn hyperaccumulating Thlaspi praecox (Brassicaceae). Plant biomass and tissue concentrations of Cd, Pb, Zn, Fe and Ni were found to vary during development, while no variation in P, K, Ca, Mn and Cu tissue concentrations were observed. The lowest Cd bioaccumulation in rosette leaves (BAF(RL)) observed during seeding was partially attributed to lower translocation from roots to rosette leaves and partially to high translocation to stalks, indicating a high Cd mobility to reproductive tissues, in line with our previous studies. The highest intensity of AM colonisation (M%) was observed in the flowering phase and was accompanied by increased root Cd, Zn, Pb and Fe contents. In addition, a positive correlation between AM colonisation and Fe contents in rosette leaves was found. The results indicate developmental dependence of AM formation, accompanied by selective changes in nutrient acquisition in T. praecox that are related to increased plant needs, and the protective role of AM colonisation on metal polluted sites during the reproductive period.

Characterization of Ni-tolerant methylobacteria associated with the hyperaccumulating plant Thlaspi goesingense and description of Methylobacterium goesingense sp. nov.

Various pink-pigmented facultative methylotrophic (PPFM) bacteria (strains iEII3, iEIV1, iEI6, iEII1, iEIII3 iEIII4, iEIII5, iRII1, iRII2, iRIII1, iRIV1 and iRIV2) were obtained from the rhizosphere and endosphere of hyperaccumulating plant Thlaspi goesingense grown in Redschlag, Austria [R. Idris, R. Trifonova, M. Puschenreiter, W.W. Wenzel, A. Sessitsch, Bacterial communities associated with flowering plants of the Ni hyperaccumulator Thlaspi goesingense, Appl. Environ. Microbiol. 70 (2004) 2667-2677]. Due to their unexpected diversity, abundance and nickel tolerance they were further characterized by detailed 16S rRNA gene analysis, DNA-DNA hybridization, fatty acid analysis, heavy metal tolerance, screening for known Ni resistance genes and phenotypic analysis. These strains were found to exhibit different multiple heavy metal resistance characteristics to Ni, Cd, Co, Zn and Cr. On the basis of their physiological and genotypic properties, strains could be grouped with Methylobacterium extorquens and M. mesophilicum. One endophyte, strain iEII3, was found to belong to a novel species for which the name M. goesingense is proposed.

Colonisation of a Zn, Cd and Pb hyperaccumulator Thlaspi praecox Wulfen with indigenous arbuscular mycorrhizal fungal mixture induces changes in heavy metal and nutrient uptake.

Plants of the Zn, Cd and Pb hyperaccumulator Thlaspi praecox Wulfen (Brassicaceae) inoculated or not with indigenous arbuscular mycorrhizal (AM) fungal mixture were grown in a highly Cd, Zn and Pb contaminated substrate in order to evaluate the functionality of symbiosis and assess the possible impact of AM colonisation on heavy metal uptake and tolerance. The results suggest AM development in the metal hyperaccumulating T. praecox is favoured at elevated nutrient demands, e.g. during the reproductive period. AM colonisation parameters positively correlated with total soil Cd and Pb. Colonised plants showed significantly improved nutrient and a decreased Cd and Zn uptake as revealed by TRXRF, thus confirming the functionality of the symbiosis. Reduced heavy metal uptake, especially at higher soil metal contents, indicates a changed metal tolerance strategy in colonised T. praecox plants. This is to our knowledge the first report on AM colonisation of the Zn, Cd and Pb hyperaccumulator T. praecox in a greenhouse experiment.

Nickel and cobalt resistance engineered in Escherichia coli by overexpression of serine acetyltransferase from the nickel hyperaccumulator plant Thlaspi goesingense.

The overexpression of serine acetyltransferase from the Ni-hyperaccumulating plant Thlaspi goesingense causes enhanced nickel and cobalt resistance in Escherichia coli. Furthermore, overexpression of T. goesingense serine acetyltransferase results in enhanced sensitivity to cadmium and has no significant effect on resistance to zinc. Enhanced nickel resistance is directly related to the constitutive overactivation of sulfur assimilation and glutathione biosynthesis, driven by the overproduction of O-acetyl-L-serine, the product of serine acetyltransferase and a positive regulator of the cysteine regulon. Nickel in the serine acetyltransferase-overexpressing strains is not detoxified by coordination or precipitation with sulfur, suggesting that glutathione is involved in reducing the oxidative damage imposed by nickel.

Zn, Cd and Pb accumulation and arbuscular mycorrhizal colonisation of pennycress Thlaspi praecox Wulf. (Brassicaceae) from the vicinity of a lead mine and smelter in Slovenia.

Significant hyperaccumulation of Zn, Cd and Pb in field samples of Thlaspi praecox Wulf. collected from a heavy metal polluted area in Slovenia was found, with maximal shoot concentrations of 14,590 mg kg(-1) Zn, 5960 mg kg(-1) Cd and 3500 mg kg(-1) Pb. Shoot/root ratios of 9.6 for Zn and 5.6 for Cd show that the metals were preferentially transported to the shoots. Shoot bioaccumulation factors exceeded total soil Cd levels 75-fold and total soil Zn levels 20-fold, further supporting the hyperaccumulation of Cd and Zn. Eighty percent of Pb was retained in roots, thus indicating exclusion as a tolerance strategy for Pb. Low level colonisation with arbuscular mycorrhizal fungi (AMF) of a Paris type was observed at the polluted site, whereas at the non-polluted site Arum type colonisation was more common. To our knowledge this is the first report of Cd hyperaccumulation and AMF colonisation in metal hyperaccumulating T. praecox.

Bacterial communities associated with flowering plants of the Ni hyperaccumulator Thlaspi goesingense.

Thlaspi goesingense is able to hyperaccumulate extremely high concentrations of Ni when grown in ultramafic soils. Recently it has been shown that rhizosphere bacteria may increase the heavy metal concentrations in hyperaccumulator plants significantly, whereas the role of endophytes has not been investigated yet. In this study the rhizosphere and shoot-associated (endophytic) bacteria colonizing T. goesingense were characterized in detail by using both cultivation and cultivation-independent techniques. Bacteria were identified by 16S rRNA sequence analysis, and isolates were further characterized regarding characteristics that may be relevant for a beneficial plant-microbe interaction-Ni tolerance, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and siderophore production. In the rhizosphere a high percentage of bacteria belonging to the Holophaga/Acidobacterium division and alpha-Proteobacteria were found. In addition, high-G+C gram-positive bacteria, Verrucomicrobia, and microbes of the Cytophaga/Flexibacter/Bacteroides division colonized the rhizosphere. The community structure of shoot-associated bacteria was highly different. The majority of clones affiliated with the Proteobacteria, but also bacteria belonging to the Cytophaga/Flexibacter/Bacteroides division, the Holophaga/Acidobacterium division, and the low-G+C gram-positive bacteria, were frequently found. A high number of highly related Sphingomonas 16S rRNA gene sequences were detected, which were also obtained by the cultivation of endophytes. Rhizosphere isolates belonged mainly to the genera Methylobacterium, Rhodococcus, and Okibacterium, whereas the majority of endophytes showed high levels of similarity to Methylobacterium mesophilicum. Additionally, Sphingomonas spp. were abundant. Isolates were resistant to Ni concentrations between 5 and 12 mM; however, endophytes generally tolerated higher Ni levels than rhizosphere bacteria. Almost all bacteria were able to produce siderophores. Various strains, particularly endophytes, were able to grow on ACC as the sole nitrogen source.

Phytoalexins from Thlaspi arvense, a wild crucifer resistant to virulent Leptosphaeria maculans: structures, syntheses and antifungal activity.

Phytoalexins are inducible chemical defenses produced by plants in response to diverse forms of stress, including microbial attack. Our search for phytoalexins from cruciferous plants resistant to economically important fungal diseases led us to examine stinkweed or pennycress (Thlaspi arvense), a potential source of disease resistance to blackleg. We have investigated phytoalexin production in leaves of T. arvense under abiotic (copper chloride) and biotic elicitation by Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.], and report here two phytoalexins, wasalexin A and arvelexin (4-methoxyindolyl-3-acetonitrile), their syntheses and antifungal activity against isolates of P. lingam/L. maculans, as well as the isolation of isovitexin, a constitutive glycosyl flavonoid of stinkweed, having antioxidant properties but devoid of antifungal activity.

Comparative 16S rDNA and 16S rRNA sequence analysis indicates that Actinobacteria might be a dominant part of the metabolically active bacteria in heavy metal-contaminated bulk and rhizosphere soil.

Bacterial diversity in 16S ribosomal DNA and reverse-transcribed 16S rRNA clone libraries originating from the heavy metal-contaminated rhizosphere of the metal-hyperaccumulating plant Thlaspi caerulescens was analysed and compared with that of contaminated bulk soil. Partial sequence analysis of 282 clones revealed that most of the environmental sequences in both soils affiliated with five major phylogenetic groups, the Actinobacteria, alpha-Proteobacteria, beta-Proteobacteria, Acidobacteria and the Planctomycetales. Only 14.7% of all phylotypes (sequences with similarities> 97%), but 45% of all clones, were common in the rhizosphere and the bulk soil clone libraries. The combined use of rDNA and rRNA libraries indicated which taxa might be metabolically active in this soil. All dominant taxa, with the exception of the Actinobacteria, were relatively less represented in the rRNA libraries compared with the rDNA libraries. Clones belonging to the Verrucomicrobiales, Firmicutes, Cytophaga-Flavobacterium-Bacteroides and OP10 were found only in rDNA clone libraries, indicating that they might not represent active constituents in our samples. The most remarkable result was that sequences belonging to the Actinobacteria dominated both bulk and rhizosphere soil libraries derived from rRNA (50% and 60% of all phylotypes respectively). Seventy per cent of these clone sequences were related to the Rubrobacteria subgroups 2 and 3, thus providing for the first time evidence that this group of bacteria is probably metabolically active in heavy metal-contaminated soil.

Colonization of pennycresses (Thlaspi spp.) of the Brassicaceae by arbuscular mycorrhizal fungi.

Members of the Brassicaceae are generally believed to be non-mycorrhizal. Pennycress (Thlaspi) species of this family from diverse locations in Slovenia, Austria, Italy and Germany were examined for their colonisation by arbuscular mycorrhizal fungi (AMF). Meadow species (T. praecox, T. caerulescens and T. montanum) were sparsely but distinctly colonised, as indicated by the occurrence of intraradical hyphae, vesicles, coils, and occasionally arbuscules. Species from other locations were poorly colonised, but arbuscules were not discernible. The genus Thlaspi comprises several heavy metal hyperaccumulating species (T. caerulescens, T. goesingense, T. calaminare, T. cepaeifolium). All samples collected from heavy metal soils were at best poorly colonized. Thus the chance is small to find a "hypersystem" in phytoremediation consisting of an AM fungus which prevents the uptake of the major part of the heavy metals and of a Thlaspi species which effectively deposits the residual heavy metals inevitably taken up into its vacuoles. In two different PCR approaches, fungal DNA was amplified from most of the Thlaspi roots examined, even from those with a very low incidence of AMF colonization. Sequencing of the 28S- and 18S-rDNA PCR-products revealed that different Thlaspi field samples were colonized by Glomus intraradices and thus by a common AM fungus. However, none of the sequences obtained was identical to any other found in the present study or deposited in the databanks, which might indicate that a species continuum exists in the G. intraradices clade. An effective colonization of Thlaspi by AMF could not be established in greenhouse experiments. Although the data show that Thlaspi can be colonized by AMF, it is doubtful whether an effective symbiosis with the mutual exchange of metabolites is formed by both partners.