Rhexocercosporidium matricariae sp. nov. - a new pathogen on the medicinal plant Matricaria recutita.

A new disease was observed on chamomile plants (Matricaria recutita) in various commercially cultivated fields in 2016 and 2017 in Germany. From symptomatic stems and leaves, the fungal species described here was isolated repeatedly. Koch´s postulates using in planta experiments were fulfilled by inoculation of chamomile plants proving the fungus to be the causal agent of the disease. Morphological studies and phylogenetic analyses using internal transcribed spacer, large subunit, and translation elongation factor-1α sequences suggested that the fungus represents a new species within the genus Rhexocercosporidium (Helotiales). The data are presented together with a description of the growth parameters, and comprehensive illustrations of the new species, Rhexocercosporidium matricariae. All species so far assigned to Rhexocercosporidium are compared and discussed. The combination Rhexocercosporidium microsporum is validated.

Glucosylation prevents plant defense activation in phloem-feeding insects.

The metabolic adaptations by which phloem-feeding insects counteract plant defense compounds are poorly known. Two-component plant defenses, such as glucosinolates, consist of a glucosylated protoxin that is activated by a glycoside hydrolase upon plant damage. Phloem-feeding herbivores are not generally believed to be negatively impacted by two-component defenses due to their slender piercing-sucking mouthparts, which minimize plant damage. However, here we document that glucosinolates are indeed activated during feeding by the whitefly Bemisia tabaci. This phloem feeder was also found to detoxify the majority of the glucosinolates it ingests by the stereoselective addition of glucose moieties, which prevents hydrolytic activation of these defense compounds. Glucosylation of glucosinolates in B. tabaci was accomplished via a transglucosidation mechanism, and two glycoside hydrolase family 13 (GH13) enzymes were shown to catalyze these reactions. This detoxification reaction was also found in a range of other phloem-feeding herbivores.

First detection of Trichothecium roseum causing leaf spots on tomato in Germany.

Tomato (Solanum lycopersicum L.) is one of the most popular vegetables in Germany and the demand for organically grown tomatoes in particular is growing steadily. In seasonal low-cost organic tomato cultivation, the leaf mould disease caused by Fulvia fulva is an increasing problem (Meyer et al. 2021). In 2020-2021, during a survey for F. fulva infected tomato leaves in Germany, untypical, circular, and dark brown necrotic spots were observed. To identify the pathogen, symptomatic leaves were collected from three different geographic locations in the north and south of Germany. Specimens were observed under a stereomicroscope and immediately processed. After surface disinfection of symptomatic leaf material, the leaf tissues were placed on potato dextrose agar (PDA) and subsequently incubated at 25 °C in the dark allowing mycelium to grow. Single spore isolates from each location were prepared for morphological and molecular analyses, as well as for pathogenicity determination. Three single spore isolates (JKI-GFP-22-010 - 012) showed moderate to rapid growth rates that reached 4 cm in diameter after 7 days. Based solely on morphology (S1), they were identified as Trichothecium roseum (Pers.) Link (Link 1809) by having pink dense mycelium with circular and rough edge colonies. The conidiophores were simple or branched (105 - 254 × 2 - 4 µm). Conidia were 2-celled, smooth, thick-walled, hyaline, ellipsoid to pyriform with slanting and truncate basal mark, and were produced in clusters, (10 - 18 × 7 - 9.5 µm). To afford the genetic data of these fungi, fungal DNA was extracted using a DNAeasy® Kit Plant Mini Kit (QIAGEN®, Germany) and then subjected to PCR using the primer sets as follows, ITS1/ITS4 (White et al. 1990) and LROR/LR6 (Vilgalys et al. 1990). The consensus sequences generated in this study were deposited in GenBank under the accession numbers ON787818 - 20 and ON787821 - 23, for ITS and LSU, respectively, and compared via NCBI-BLAST. Initial BLAST searches of both ITS and LSU regions revealed that the isolates share 99 - 100% homology with T. roseum. The morphology and phylogeny (S2) supported assignment of all isolates to T. roseum. To fulfil Koch's postulate, the pathogenicity of selected isolates (JKI-GFP-22-010 and 012) were tested by spraying spore suspensions (105 conidia ml-1) on adaxial surface of 21-day-old tomato plants cv. 'Moneymaker' (n=10). Plants sprayed with sterile water served as controls. All tomato plants were grown under greenhouse conditions (average 25 °C, 98% relative humidity). After 21 days, symptoms were identical to those of the originally infected leaves associated with T. roseum. As the disease progressed, necrotic lesions reached approximately 35 mm in diameter. The fungus was re-isolated and identified as T. roseum. No symptoms developed in the control plants. Trichothecium roseum has been detected infecting fruit, particularly on tomatoes, e.g. in Argentina (Dal et al. 2008), Brazil (Inácio et al. 2011), Korea (Yun et al. 2013), and Pakistan (Hamid et al. 2014). To our knowledge, this is the first report of T. roseum infecting tomato in Germany. As the pathogen was found in several locations in the south and north, it is likely already well established in Germany. To date, it seems to play a minor role in pathogenicity of tomatoes but should be monitored, especially with regard to climate change as there are now several recent reports from the temperate region. The disease development and interaction between susceptible host and pathogen should be concerned.

A Novel Group of Rhizobium tumorigenes-Like Agrobacteria Associated with Crown Gall Disease of Rhododendron and Blueberry.

Crown gall is an economically important and widespread plant disease caused by tumorigenic bacteria that are commonly affiliated within the genera Agrobacterium, Allorhizobium, and Rhizobium. Although crown gall disease was reported to occur on rhododendron, literature data regarding this disease are limited. In this study, an atypical group of tumorigenic agrobacteria belonging to the genus Rhizobium was identified as a causative agent of crown gall on rhododendron. Genome analysis suggested that tumorigenic bacteria isolated from rhododendron tumors are most closely related to Rhizobium tumorigenes, a new tumorigenic bacterium discovered recently on blackberry in Serbia. However, R. tumorigenes and novel rhododendron strains belong to separate species and form a homogenous clade within the genus Rhizobium, which we named the "tumorigenes" clade. Moreover, tumorigenic bacteria isolated from rhododendron seem to carry a distinct tumor-inducing (Ti) plasmid, compared with those carried by R. tumorigenes strains and Ti plasmids described thus far. To facilitate rapid identification of bacteria belonging to the "tumorigenes" clade, regardless of whether they are pathogenic or not, a conventional PCR method targeting putative chromosomal gene-encoding flagellin protein FlaA was developed in this study. Finally, our results suggested that this novel group of tumorigenic agrobacteria occurs on blueberry but it cannot be excluded that it is distributed more widely.

Powdery mildew of Chrysanthemum × morifolium: phylogeny and taxonomy in the context of Golovinomyces species on Asteraceae hosts.

The taxonomic history of the common powdery mildew of Chrysanthemum × morifolium (chrysanthemum, florist's daisy), originally described in Germany as Oidium chrysanthemi, is discussed. The position of O. chrysanthemi was investigated on the basis of morphological traits and molecular phylogenetic analyses. Based on the results of this study, this species, which is closely related to Golovinomyces artemisae, was reassessed and reallocated to Golovinomyces. The phylogenetic analysis and taxonomic reassessment of the chrysanthemum powdery mildew is supplemented by a morphological description, a summary of its worldwide distribution data, and a brief discussion of the introduction of this fungus to North America. G. chrysanthemi differs from true G. artemisiae in that it has much longer conidiophores, is not constricted at the base, and has much larger and most importantly longer conidia. The close affinity of Golovinomyces to Artemisia and Chrysanthemum species signifies a coevolutionary event between the powdery mildews concerned and their host species in the subtribe Artemisiinae (Asteraceae tribe Anthemideae). This conclusion is fully supported by the current phylogeny and taxonomy of the host plant genera and the coevolution that occurred with the host and pathogen. The following powdery mildew species, which are associated with hosts belonging to the tribe Anthemideae of the Asteraceae, are epitypified: Alphitomorpha depressa ß artemisiae (≡ Alphitomorpha artemisiae), Erysiphe artemisiae, and Oidium chrysanthemi. Erysiphe macrocarpa is neotypified. Their sequences were retrieved from the epitype collections and have been added to the phylogenetic tree. Golovinomyces orontii, an additional powdery mildew species on Chrysanthemum ×morifolium, is reported. This species is rarely found as a spontaneous infection and was obtained from inoculation experiments.

Implication of Bemisia tabaci heat shock protein 70 in Begomovirus-whitefly interactions.

The whitefly Bemisia tabaci (Gennadius) is a major cosmopolitan pest capable of feeding on hundreds of plant species and transmits several major plant viruses. The most important and widespread viruses vectored by B. tabaci are in the genus Begomovirus, an unusual group of plant viruses owing to their small, single-stranded DNA genome and geminate particle morphology. B. tabaci transmits begomoviruses in a persistent circulative nonpropagative manner. Evidence suggests that the whitefly vector encounters deleterious effects following Tomato yellow leaf curl virus (TYLCV) ingestion and retention. However, little is known about the molecular and cellular basis underlying these coevolved begomovirus-whitefly interactions. To elucidate these interactions, we undertook a study using B. tabaci microarrays to specifically describe the responses of the transcriptomes of whole insects and dissected midguts following TYLCV acquisition and retention. Microarray, real-time PCR, and Western blot analyses indicated that B. tabaci heat shock protein 70 (HSP70) specifically responded to the presence of the monopartite TYLCV and the bipartite Squash leaf curl virus. Immunocapture PCR, protein coimmunoprecipitation, and virus overlay protein binding assays showed in vitro interaction between TYLCV and HSP70. Fluorescence in situ hybridization and immunolocalization showed colocalization of TYLCV and the bipartite Watermelon chlorotic stunt virus virions and HSP70 within midgut epithelial cells. Finally, membrane feeding of whiteflies with anti-HSP70 antibodies and TYLCV virions showed an increase in TYLCV transmission, suggesting an inhibitory role for HSP70 in virus transmission, a role that might be related to protection against begomoviruses while translocating in the whitefly.

Neisserial Omp85 protein is selectively recognized and assembled into functional complexes in the outer membrane of human mitochondria.

As a consequence of their bacterial origin, mitochondria contain ß-barrel proteins in their outer membrane (OMM). These proteins require the translocase of the outer membrane (TOM) complex and the conserved sorting and assembly machinery (SAM) complex for transport and integration into the OMM. The SAM complex and the ß-barrel assembly machinery (BAM) required for biogenesis of ß-barrel proteins in bacteria are evolutionarily related. Despite this homology, we show that bacterial ß-barrel proteins are not universally recognized and integrated into the OMM of human mitochondria. Selectivity exists both at the level of the TOM and the SAM complex. Of all of the proteins we tested, human mitochondria imported only ß-barrel proteins originating from Neisseria sp., and only Omp85, the central component of the neisserial BAM complex, integrated into the OMM. PorB proteins from different Neisseria, although imported by the TOM, were not recognized by the SAM complex and formed membrane complexes only when functional Omp85 was present at the same time in mitochondria. Omp85 alone was capable of integrating other bacterial ß-barrel proteins in human mitochondria, but could not substitute for the function of its mitochondrial homolog Sam50. Thus, signals and machineries for transport and assembly of ß-barrel proteins in bacteria and human mitochondria differ enough to allow only a certain type of ß-barrel proteins to be targeted and integrated in mitochondrial membranes in human cells.

Phylogeny and taxonomy of the genera of Erysiphaceae, part 2: Neoerysiphe.

The second contribution to a new series devoted to the phylogeny and taxonomy of powdery mildews is presented. An overview of Neoerysiphe species is given, including references to ex-type sequences or, if unavailable, representative reference sequences for phylogenetic-taxonomic purposes are provided. The new species N. stachydis is described, and Striatoidium jaborosae is reduced to synonymy with Neoerysiphe macquii. Epitypes with ex-epitype sequences are designated for Alphitomorpha ballotae, A. labiatarum, Erysiphe galii, E. chelones, and E. galeopsidis. Based on phylogenetic analyses, it has been demonstrated that Neoerysiphe cumminsiana is confined to its type host, Roldana hartwegii (= Senecio seemannii), and other North and South American parasites on Asteraceae hosts, previously assigned to this species, pertain to N. macquii. The first record of N. macquii from Europe (Germany) on cultivated Bidens aurea was confirmed by sequencing. Sequence analysis of type material of N. rubiae reveals that this species should be excluded from Neoerysiphe; however, the true affinity of this taxon is not yet clear.

Phylogeny and taxonomy of powdery mildew caused by Erysiphe species on Lupinus hosts.

The genus Lupinus (Fabaceae) consists of over 250 plant species located throughout the world. Powdery mildew, caused by Erysiphe species, is a common disease infecting these ecologically, ornamentally, and agriculturally important plants. In the present work, we conducted phylogenetic and taxonomic analyses on Erysiphe species colonizing hosts of the leguminous genus Lupinus, using sequences from the internal transcribed spacer (ITS) and 28S genomic regions. Powdery mildews of the genus Erysiphe on Fabaceae are taxonomically intricate and challenging. Therefore, it is necessary to phylogenetically analyze the DNA retrieved from powdery mildew on lupines in a broad context that includes common and allied powdery mildew species that occur on a range of leguminous plants such as Erysiphe astragali, E. baeumleri, E. pisi, and E. trifoliorum. A new species Erysiphe lupini, found in the USA on Lupinus lepidus, L. polyphyllus, and Lupinus sp., is described. Additionally, Erysiphe intermedia (≡ Microsphaera trifolii var. intermedia) has been confirmed as a North American lupine powdery mildew that is a sister species to E. astragali on Astragalus spp. European Erysiphe collections on lupines were often referred to as E. intermedia, but our analyses have shown that they pertain to E. trifoliorum. The E. trifoliorum clade is composed of several species (i.e. E. baeumleri, E. euonymi, E. hyperici, and E. trifoliorum), that cannot be sufficiently resolved based solely on ITS+28S sequences. Morphological and biological differences between the species are discussed and provide evidence that the species concerned should be maintained. Finally, a sequence obtained from a powdery mildew collected in Portugal on the native Lupinus micranthus pertained to the Erysiphe guarinonii clade. This collection is tentatively treated as Erysiphe sp. To fix the application of the species names E. astragali, E. baeumleri (including its synonym E. marchica), and E. intermedia, epitypes have been designated with ex-epitype sequences.

Phylogeny and taxonomy of Erysiphe spp. on Rhododendron, with a special emphasis on North American species.

The genus Rhododendron comprises over 1000 evergreen and deciduous species. In the Pacific Northwest Coast region of North America (PNWC), powdery mildews infecting deciduous Rhododendron spp. are well documented but less so on evergreen Rhododendron spp. Infections of both groups of hosts historically have been attributed to Erysiphe azaleae or E. vaccinii. No formal characterizations of powdery mildew fungi infecting either deciduous or evergreen Rhododendron spp. in the PNWC have been completed. The objectives of this study were to identify the powdery mildew pathogens infecting evergreen Rhododendron spp. in the PNWC and to assess the phylogenetic position of these fungi within the Erysiphaceae. To ascertain valid taxonomic conclusions, and to determine whether potential introductions of exotic Rhododendron powdery mildews in North America have occurred, it was necessary to put the new North American phylogenetic data into a worldwide context. Therefore, available phylogenetic data from all Erysiphe spp. on Rhododendron have been included in our analyses.Based on analyses of numerous new internal transcribed spacer (ITS) and 28S rDNA sequences and already available sequences deposited in GenBank retrieved from evergreen and deciduous Rhododendron spp., the following Erysiphe spp. could be phylogenetically confirmed (all belonging to Erysiphe sect. Microsphaera): Erysiphe azaleae nom. cons. (Oidium ericinum could be verified as a synonym), E. digitata (holotype sequenced), E. izuensis, and E. vaccinii. Erysiphe azaleae and E. vaccinii are epitypified with sequenced specimens, and an ex-neotype sequence has been obtained for Oidium ericinum. Erysiphe rhododendri (Erysiphe sect. Erysiphe), only known from two collections in India (Himalayan region), was not available for phylogentic analyses.

A Non-Destructive High-Speed Procedure to Obtain DNA Barcodes from Soft-Bodied Insect Samples with a Focus on the Dipteran Section of Schizophora.

While the need for biodiversity research is growing, paradoxically, global taxonomical expertise is decreasing as a result of the neglected funding for young academics in taxonomy. Non-destructive approaches for DNA barcoding are necessary for a more efficient use of this dwindling expertise to fill gaps, and identify incorrect entries in sequence databases like BOLD or GenBank. They are efficient because morphological re-examination of species vouchers is still possible post-DNA barcoding. Non-destructive approaches for Diptera with a comprehensive species representation or the consideration of diagnostic fragile morphological characters are missing. Additionally, most non-destructive approaches combine a time intensive and non-destructive digestion step with common DNA extraction methods, such as commercial kits or CTAB DNA isolation. We circumvented those approaches and combined a modified non-destructive TE buffer high-speed DNA extraction, with a PCR inhibitor-resistant PCR reaction system, to a non-destructive DNA barcoding procedure for fresh and frozen samples of the Schizophora (Diptera). This method avoids morphological impairment and the application of harmful chemicals, is cost and time effective, restricts the need for laboratory equipment to a minimum, and prevents cross-contamination risk during DNA isolation. Moreover, the study indicates that the presented non-destructive DNA barcoding procedure is transferable to other soft-bodied insects. We suggest that PCR inhibitor-resistant master mixes enable the development of new-and the modification of existing-non-destructive approaches with the avoidance of further DNA template cleaning.

Phylogeny and taxonomy of Erysiphe berberidis (s. lat.) revisited.

Phylogenetic and morphological analyses have been conducted on powdery mildew specimens on different Berberis and Mahonia spp. from Asia, Europe and North America. The present study showed that collections of Erysiphe berberidis exhibit a high degree of morphological plasticity of the sexual morph, in contrast to their morphologically, rather uniform, asexual morph. In phylogenetic tree, all sequences cluster in a large strongly supported clade, without any indication and support for further differentiation into cryptic species. There are three morphological types within E. berberidis s. lat. that contain consistent differences. Until future multi-locus analyses will be available, we prefer to treat these 'morphological types' as varieties. These include Erysiphe berberidis var. berberidis, E. berberidis var. asiatica, and E. berberidis var. dimorpha comb. nov. (≡ Microsphaera berberidis var. dimorpha, M. berberidicola, and M. multappendicis). To fix the application of species name E. berberidis, an appropriate epitype was designated, with an ITS sequences.

Phylogeny and taxonomy of Podosphaera filipendulae (Erysiphaceae) revisited.

The phylogeny and taxonomy of Podosphaera filipendulae (including P. filipendulensis, syn. nov.) have been examined. Asian, European and North American collections were examined and the nucleotides sequences of their partial rDNA region were determined. In particular, the relationship between P. filipendulae and P. spiraeae was analysed. The results confirmed P. filipendulae and P. spiraeae as two separate, morphologically similar species. The phylogenetic analysis revealed a similar phylogeny to that of the host genera. Although ITS sequences retrieved from Asian, European and North American specimens of P. filipendulae on various Filipendula spp. are identical to sequences from P. macularis on hop, there is consistently one base substitution at the 5'-end of 28S rRNA gene between the species. This result provides evidence that the hop powdery mildew and P. filipendulae are biologically and morphologically clearly distinguished, and should be maintained as two separate species.

Identification of a Sulfatase that Detoxifies Glucosinolates in the Phloem-Feeding Insect Bemisia tabaci and Prefers Indolic Glucosinolates.

Cruciferous plants in the order Brassicales defend themselves from herbivory using glucosinolates: sulfur-containing pro-toxic metabolites that are activated by hydrolysis to form compounds, such as isothiocyanates, which are toxic to insects and other organisms. Some herbivores are known to circumvent glucosinolate activation with glucosinolate sulfatases (GSSs), enzymes that convert glucosinolates into inactive desulfoglucosinolates. This strategy is a major glucosinolate detoxification pathway in a phloem-feeding insect, the silverleaf whitefly Bemisia tabaci, a serious agricultural pest of cruciferous vegetables. In this study, we identified and characterized an enzyme responsible for glucosinolate desulfation in the globally distributed B. tabaci species MEAM1. In in vitro assays, this sulfatase showed a clear preference for indolic glucosinolates compared with aliphatic glucosinolates, consistent with the greater representation of desulfated indolic glucosinolates in honeydew. B. tabaci might use this detoxification strategy specifically against indolic glucosinolates since plants may preferentially deploy indolic glucosinolates against phloem-feeding insects. In vivo silencing of the expression of the B. tabaci GSS gene via RNA interference led to lower levels of desulfoglucosinolates in honeydew. Our findings expand the knowledge on the biochemistry of glucosinolate detoxification in phloem-feeding insects and suggest how detoxification pathways might facilitate plant colonization in a generalist herbivore.

Green fluorescent protein (GFP) as a reporter gene for the plant pathogenic oomycete Phytophthora ramorum.

Transgenic Phytophthora ramorum strains that produce green fluorescent protein (GFP) constitutively were obtained after stable DNA integration using a polyethylene glycol and CaCl2-based transformation protocol. Green fluorescent protein production was studied in developing colonies and in different propagules of the pathogen to evaluate its use in molecular and physiological studies. About 12% of the GFP transformants produced GFP to a level detectable by a confocal laser scanning microscope. Green fluorescent protein could be visualized in structures with vital protoplasm, such as hyphal tips and germinating cysts. In infection studies with Rhododendron, one of the GFP expressing strains showed aggressiveness equal to that of the corresponding non-labelled isolate. Thus, GFP could be used as a reporter gene in P. ramorum. Limitations of the technology are discussed.

Effects of site and plant species on rhizosphere community structure as revealed by molecular analysis of microbial guilds.

The bacterial and fungal rhizosphere communities of strawberry (Fragaria ananassa Duch.) and oilseed rape (Brassica napus L.) were analysed using molecular fingerprints. We aimed to determine to what extent the structure of different microbial groups in the rhizosphere is influenced by plant species and sampling site. Total community DNA was extracted from bulk and rhizosphere soil taken from three sites in Germany in two consecutive years. Bacterial, fungal and group-specific (Alphaproteobacteria, Betaproteobacteria and Actinobacteria) primers were used to PCR-amplify 16S rRNA and 18S rRNA gene fragments from community DNA prior to denaturing gradient gel electrophoresis (DGGE) analysis. Bacterial fingerprints of soil DNA revealed a high number of equally abundant faint bands, while rhizosphere fingerprints displayed a higher proportion of dominant bands and reduced richness, suggesting selection of bacterial populations in this environment. Plant specificity was detected in the rhizosphere by bacterial and group-specific DGGE profiles. Different bulk soil community fingerprints were revealed for each sampling site. The plant species was a determinant factor in shaping similar actinobacterial communities in the strawberry rhizosphere from different sites in both years. Higher heterogeneity of DGGE profiles within soil and rhizosphere replicates was observed for the fungi. Plant-specific composition of fungal communities in the rhizosphere could also be detected, but not in all cases. Cloning and sequencing of 16S rRNA gene fragments obtained from dominant DGGE bands detected in the bacterial profiles of the Rostock site revealed that Streptomyces sp. and Rhizobium sp. were among the dominant ribotypes in the strawberry rhizosphere, while sequences from Arthrobacter sp. corresponded to dominant bands from oilseed rape bacterial fingerprints.

The rhizosphere effect on bacteria antagonistic towards the pathogenic fungus Verticillium differs depending on plant species and site.

Rhizobacteria with antagonistic activity towards plant pathogens play an essential role in root growth and plant health and are influenced by plant species in their abundance and composition. To determine the extent of the effect of the plant species and of the site on the abundance and composition of bacteria with antagonistic activity towards Verticillium dahliae, bacteria isolated from the rhizosphere of two Verticillium host plants, oilseed rape and strawberry, and from bulk soil were analysed at three different locations in Germany over two growing seasons. A total of 6732 bacterial isolates screened for in vitro antagonism towards Verticillium resulted in 560 active isolates, among which Pseudomonas (77%) and Serratia (6%) were the most dominant genera. The rhizosphere effect on the antagonistic bacterial community was shown by an enhanced proportion of antagonistic isolates, by enrichment of specific amplified ribosomal DNA restriction analysis types, species and genotypes, and by a reduced diversity in the rhizosphere in comparison to bulk soil. Such an effect was influenced by the plant species and by the site of its cultivation. Altogether, 16S rRNA gene sequencing of 66 isolates resulted in the identification of 22 different species. Antagonists of the genus Serratia were preferentially isolated from oilseed rape rhizosphere, with the exception of one site. For isolates of Pseudomonas and Serratia, plant-specific and site-specific genotypes were found.

Survival of gfp-tagged antagonistic bacteria in the rhizosphere of tomato plants and their effects on the indigenous bacterial community.

The survival and colonization patterns of Pseudomonas putida PRD16 and Enterobacter cowanii PRF116 in the rhizosphere of greenhouse-grown tomato plants and the effects of their inoculation on the indigenous bacterial community were followed by selective plating, molecular fingerprinting, and confocal laser scanning microscopy (CLSM) over 3 weeks. Both strains, which showed in vitro antagonistic activity against Ralstonia solanacearum, were previously tagged with gfp. Seed and root inoculation were compared. Although plate counts decreased for both gfp-tagged antagonists, PRD16 showed a better survival in the rhizosphere of tomato roots independent of the inoculation method. Analysis of 16S rRNA gene fragments amplified from total community DNA by denaturing gradient gel electrophoresis and CLSM confirmed the decrease in the relative abundance of the inoculant strains. Pronounced differences in the Pseudomonas community patterns for plants inoculated with PRD16 compared to the control were detected 3 weeks after root inoculation, indicating a longer-lasting effect. Analysis by CLSM showed rather heterogeneous colonization patterns for both inoculant strains. In comparison with seed inoculation, root inoculation led to a much better colonization as evidenced by all three methods. The colonization patterns observed by CLSM provide important information on the sampling strategy required for monitoring inoculant strains in the rhizosphere.

Fungal endophytes in potato roots studied by traditional isolation and cultivation-independent DNA-based methods.

The composition and relative abundance of endophytic fungi in roots of field-grown transgenic T4-lysozyme producing potatoes and the parental line were assessed by classical isolation from root segments and cultivation-independent techniques to test the hypothesis that endophytic fungi are affected by T4-lysozyme. Fungi were isolated from the majority of root segments of both lines and at least 63 morphological groups were obtained with Verticillium dahliae, Cylindrocarpon destructans, Colletotrichum coccodes and Plectosporium tabacinum as the most frequently isolated species. Dominant bands in the fungal fingerprints obtained by denaturing gradient gel electrophoresis analysis of 18S rRNA gene fragments amplified from total community DNA corresponded to the electrophoretic mobility of the 18S rRNA gene fragments of the three most abundant fungal isolates, V. dahliae, C. destructans and Col. coccodes, but not to P. tabacinum. The assignment of the bands to these isolates was confirmed for V. dahliae and Col. coccodes by sequencing of clones. Verticillium dahliae was the most abundant endophytic fungus in the roots of healthy potato plants. Differences in the relative abundance of endophytic fungi colonizing the roots of T4-lysozyme producing potatoes and the parental line could be detected by both methods.

The contribution of translesion synthesis polymerases on geminiviral replication.

Geminiviruses multiply primarily in the plant phloem, but never in meristems. Their Rep protein can activate DNA synthesis in differentiated cells. However, when their single-stranded DNA is injected into the phloem by insects, no Rep is present for inducing initial complementary strand replication. Considering a contribution of translesion synthesis (TLS) polymerases in plants, four of them (Polη, Polζ, Polκ, Rev1) are highly and constitutively expressed in differentiated tissues like the phloem. Two geminiviruses (Euphorbia yellow mosaic virus, Cleome leaf crumple virus), inoculated either biolistically or by whiteflies, replicated in Arabidopsis thaliana mutant lines of these genes to the same extent as in wild type plants. Comparative deep sequencing of geminiviral DNAs, however, showed a high exchange rate (10(-4)-10(-3)) similar to the phylogenetic variation described before and a significant difference in nucleotide substation rates if Polη and Polζ were absent, with a differential response to the viral DNA components.