Wechselwirkung zwischen Maiswurzel-assoziierten Mikroorganismen und dem Western Corn Rootworm

The Western Corn Rootworm (WCR, Diabrotica virgifera virgifera LeConte) is an important maize pest in North America and Europe. This thesis aimed (i) to investigate the effect of the root larval feeding on the microbial community in the rhizosphere of four maize genotypes grown in three different soil types (chapter 3); (ii) to study the complex interactions among WCR, Glomus intraradices (G.i.) and microbial communities in the rhizosphere and endorhiza of maize plants (chapter 4); (iii) to investigate the effect of the soil type on the fungal and bacterial communities inhabiting the eggs and the digestive tract of WCR larvae (chapter 5). The research described in chapter 3 revealed that the root feeding of WCR larvae increased the abundance of the bacterial population Acinetobacter calcoaceticus.In chapter 4 has been shown that G.i. reduces the WCR larval development. Shifts of the bacterial and fungal community composition in the rhizosphere and in root of G.i.-treated plants were observed, suggesting that G.i could contribute to the control of WCR larvae either directly or indirectly through shifts in the endophytic microbial communities via plant-mediated mechanisms.In chapter 5 we demonstrated that the soil type does not influence bacterial and fungal communities in the gut of WCR larvae. Relatively simple microbial communities dominated the WCR gut: Fusarium spp. and Gibberella zeae were dominant within the fungi, while Wolbachia sp. and Herbaspirillum sp. were dominant within the bacteria. In conclusion, the results acquired in this thesis provided additional insight into the multitrophic interaction among WCR larvae and rhizospheric- and root-associated microorganisms of maize plants.Der Western Corn Rootworm (WCR) (Diabrotica virgifera virgifera LeConte) ist ein bedeutender Maisschädling in Nordamerika und Europa. Die Ziele dieser Dissertation waren (i) Untersuchung des Effektes des Wurzellarvenfraßes auf die mikrobielle Gemeinschaft in der Rhizosphäre von vier Maisgenotypen in drei unterschiedlichen Bodentypen; (ii) Erforschung der komplexen Interaktion zwischen WCR, Glomus intraradices (G.i.) und der mikrobiellen Gemeinschaft der Rhizosphäre und Endorhiza der Maispflanzen; (iii) Analyse des Effektes des Bodentyps auf Pilz- und bakterielle Gemeinschaften in den Eiern und in dem Verdauungstrakt der WCR-Larven. Es konnte ein deutlicher Effekt des Wurzelfraßes der WCR-Larven auf die Abundanz der bakteriellen Population Acinetobacter calcoaceticus nachgewiesen werden. Im Gegensatz dazu wurden keine Effekte des Larvenfraßes auf die endophytische mikrobielle Population beobachtet. Weiterhin wurde gezeigt das G.i. die Entwicklung der WCR-Larven reduziert. Da Veränderungen der Zusammensetzung der mikrobiellen Gemeinschaften in der Rhizosphäre und in G.i.-behandelten Pflanzen beobachtet wurden, könnte G.i. an einer direkten oder indirekten Kontrolle der WCR-Larven durch Veränderungen in der endophytischen mikrobiellen Gemeinschaft durch pflanzenvermittelte Mechanismen beteiligt sein. Interessanterweise wurde kein Einfluss des Bodentyps auf die mikrobielle Gemeinschaft im Darm der WCR-Larven beobachtet. Der Darm des WCR wird von verhältnismäßig einfachen mikrobiellen Gemeinschaften besiedelt: unter den Pilzen waren Fusarium spp. und Gibberella zeae dominant, während hingegen Wolbachia sp. und Herbaspirillum sp. dominante bakterielle Arten darstellten. Diese Studie dient dem Verständnis der multitrophen Wechselwirkung zwischen WCR-Larven und rhizosphären- und wurzel-assoziierten Mikroorganismen der Maispflanze

Multitrophic interactions among Western Corn Rootworm, Glomus intraradices and microbial communities in the rhizosphere and endorhiza of maize

The complex interactions among the maize pest Western Corn Rootworm (WCR), Glomus intraradices (GI-recently renamed Rhizophagus intraradices) and the microbial communities in both rhizosphere and endorhiza of maize have been investigated in view of new pest control strategies. In a greenhouse experiment, different maize treatments were established: C (control plants), W (plants inoculated with WCR), G (plants inoculated with GI), GW (plants inoculated with GI and WCR). After 20 days of WCR root feeding, larval fitness was measured. Dominant arbuscular mycorrhizal fungi (AMF) in soil and maize endorhiza were analyzed by cloning of 18S rRNA gene fragments of AMF, restriction fragment length polymorphism and sequencing. Bacterial and fungal communities in the rhizosphere and endorhiza were investigated by denaturing gradient gel electrophoresis of 16S rRNA gene and ITS fragments, PCR amplified from total community DNA, respectively. GI reduced significantly WCR larval development and affected the naturally occurring endorhiza AMF and bacteria. WCR root feeding influenced the endorhiza bacteria as well. GI can be used in integrated pest management programs, rendering WCR larvae more susceptible to predation by natural enemies. The mechanisms behind the interaction between GI and WCR remain unknown. However, our data suggested that GI might act indirectly via plant-mediated mechanisms influencing the endorhiza microbial communities

Correlation between the genomic o454-nlpD region polymorphisms, virulence gene equipment and phylogenetic group of extraintestinal Escherichia coli (ExPEC) enables pathotyping irrespective of host, disease and source of isolation

Background: The mutS-rpoS intergenic region in E. coli displays a mosaic structure which revealed pathotype specific patterns. To assess the importance of this region as a surrogate marker for the identification of highly virulent extraintestinal pathogenic E. coli (ExPEC) strains we aimed to: (i) characterize the genetic diversity of the mutS gene and the o454-nlpD genomic region among 510 E. coli strains from animals and humans; (ii) delineate associations between the polymorphism of this region and features such as phylogenetic background of E. coli, pathotype, host species, clinical condition, serogroup and virulence associated genes (VAG)s; and (iii) identify the most important VAGs for classification of the o454-nlpD region. Methods: Size variation in the o454-nlpD region was investigated by PCR amplification and sequencing. Phylogenetic relationships were assessed by Ecor- and Multilocus sequence- typing (MLST), and a comparative analysis between mutS gene phylogenetic tree obtained with RAxML and the MLST grouping method was performed. Correlation between o454-nlpD patterns and the features described above were analysed. In addition, the importance of 47 PCR-amplified ExPEC-related VAGs for classification of o454-nlpD patterns was investigated by means of Random Forest algorithm. Results: Four main structures (patterns I-IV) of the o454-nlpD region among ExPEC and commensal E. coli strains were identified. Statistical analysis showed a positive and exclusive association between pattern III and the ExPEC strains. A strong association between pattern III and either the Ecor group B2 or the sequence type complexes known to represent the phylogenetic background of highly virulent ExPEC strains (such as STC95, STC73 and STC131) was found as well. RF analyses determined five genes (csgA, malX, chuA, sit, and vat) to be suitable to predict pattern III strains. Conclusion: The significant association between pattern III and group B2 strains suggested the o454-nlpD region to be of great value in identifying highly virulent strains among the mixed population of E. coli promising to be the basis of a future typing tool for ExPEC and their gut reservoir. Furthermore, top-ranked VAGs for classification and prediction of pattern III were identified. These data are most valuable for defining ExPEC pathotype in future in vivo assays

Multitrophic Interaction in the Rhizosphere of Maize: Root Feeding of Western Corn Rootworm Larvae Alters the Microbial Community Composition

BACKGROUND: Larvae of the Western Corn Rootworm (WCR) feeding on maize roots cause heavy economical losses in the US and in Europe. New or adapted pest management strategies urgently require a better understanding of the multitrophic interaction in the rhizosphere. This study aimed to investigate the effect of WCR root feeding on the microbial communities colonizing the maize rhizosphere. METHODOLOGY/PRINCIPAL FINDINGS: In a greenhouse experiment, maize lines KWS13, KWS14, KWS15 and MON88017 were grown in three different soil types in presence and in absence of WCR larvae. Bacterial and fungal community structures were analyzed by denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA gene and ITS fragments, PCR amplified from the total rhizosphere community DNA. DGGE bands with increased intensity were excised from the gel, cloned and sequenced in order to identify specific bacteria responding to WCR larval feeding. DGGE fingerprints showed that the soil type and the maize line influenced the fungal and bacterial communities inhabiting the maize rhizosphere. WCR larval feeding affected the rhiyosphere microbial populations in a soil type and maize line dependent manner. DGGE band sequencing revealed an increased abundance of Acinetobacter calcoaceticus in the rhizosphere of several maize lines in all soil types upon WCR larval feeding. CONCLUSION/SIGNIFICANCE: The effects of both rhizosphere and WCR larval feeding seemed to be stronger on bacterial communities than on fungi. Bacterial and fungal community shifts in response to larval feeding were most likely due to changes of root exudation patterns. The increased abundance of A. calcoaceticus suggested that phenolic compounds were released upon WCR wounding

Metabolic phenotype of clinical and environmental Mycobacterium avium subsp. hominissuis isolates

Background Mycobacterium avium subsp. hominissuis (MAH) is an emerging opportunistic human pathogen. It can cause pulmonary infections, lymphadenitis and disseminated infections in immuno-compromised patients. In addition, MAH is widespread in the environment, since it has been isolated from water, soil or dust. In recent years, knowledge on MAH at the molecular level has increased substantially. In contrast, knowledge of the MAH metabolic phenotypes remains limited. Methods In this study, for the first time we analyzed the metabolic substrate utilization of ten MAH isolates, five from a clinical source and five from an environmental source. We used BIOLOG Phenotype MicroarrayTM technology for the analysis. This technology permits the rapid and global analysis of metabolic phenotypes. Results The ten MAH isolates tested showed different metabolic patterns pointing to high intra-species diversity. Our MAH isolates preferred to use fatty acids such as Tween, caproic, butyric and propionic acid as a carbon source, and L-cysteine as a nitrogen source. Environmental MAH isolates resulted in being more metabolically active than clinical isolates, since the former metabolized more strongly butyric acid (p = 0.0209) and propionic acid (p = 0.00307). Discussion Our study provides new insight into the metabolism of MAH. Understanding how bacteria utilize substrates during infection might help the developing of strategies to fight such infections

Microbial Communities Associated with the Larval Gut and Eggs of the Western Corn Rootworm

<div><h3>Background</h3><p>The western corn rootworm (WCR) is one of the economically most important pests of maize. A better understanding of microbial communities associated with guts and eggs of the WCR is required in order to develop new pest control strategies, and to assess the potential role of the WCR in the dissemination of microorganisms, e.g., mycotoxin-producing fungi.</p> <h3>Methodology/Principal Findings</h3><p>Total community (TC) DNA was extracted from maize rhizosphere, WCR eggs, and guts of larvae feeding on maize roots grown in three different soil types. Denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rRNA gene and ITS fragments, PCR-amplified from TC DNA, were used to investigate the fungal and bacterial communities, respectively. Microorganisms in the WCR gut were not influenced by the soil type. Dominant fungal populations in the gut were affiliated to <em>Fusarium</em> spp., while <em>Wolbachia</em> was the most abundant bacterial genus. Identical ribosomal sequences from gut and egg samples confirmed a transovarial transmission of <em>Wolbachia</em> sp. Betaproteobacterial DGGE indicated a stable association of <em>Herbaspirillum</em> sp. with the WCR gut. Dominant egg-associated microorganisms were the bacterium <em>Wolbachia</em> sp. and the fungus <em>Mortierella gamsii.</em></p> <h3>Conclusion/Significance</h3><p>The soil type-independent composition of the microbial communities in the WCR gut and the dominance of only a few microbial populations suggested either a highly selective environment in the gut lumen or a high abundance of intracellular microorganisms in the gut epithelium. The dominance of <em>Fusarium</em> species in the guts indicated WCR larvae as vectors of mycotoxin-producing fungi. The stable association of <em>Herbaspirillum</em> sp. with WCR gut systems and the absence of corresponding sequences in WCR eggs suggested that this bacterium was postnatally acquired from the environment. The present study provided new insights into the microbial communities associated with larval guts and eggs of the WCR. However, their biological role remains to be explored.</p> </div

Bacterial DGGE fingerprints obtained from rhizosphere samples of maize plants grown in Haplic Chernozem (Rh-HC), gut samples of larvae collected from the soil Haplic Chernozem (G-HC) and surface sterilized egg samples (E).

<p>St: ITS standard. Arrows indicate bands for which cloned ITS fragments with the same electrophoretic mobilities were sequenced (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044685#pone-0044685-t001" target="_blank">Table 1</a>). Band 1: <i>Mortierella gamsii</i>; band 2: <i>Fusarium</i> sp.; band 3: <i>Cylindrocarpon olidum</i>; band 4: <i>Thrichocladium asperum;</i> band a: <i>Candida sake</i>; band c: <i>Fusarium</i> sp.; band d: <i>Gibberella zeae</i>; band e: <i>Verticillium dahliae</i>.</p

Bacterial species identified in the larval gut and/or in the eggs of the WCR, accession numbers of 16S gene fragment sequences obtained by cloning of specific DGGE bands from gut and egg fingerprints, and bands source.

<p>Bacterial species identified in the larval gut and/or in the eggs of the WCR, accession numbers of 16S gene fragment sequences obtained by cloning of specific DGGE bands from gut and egg fingerprints, and bands source.</p

Fungal DGGE profiles showing the fungal community structure in the rhizosphere of maize plants grown in Haplic Chernozem (Rh-HC) and in the gut of WCR larvae feeding on maize roots grown in Haplic Chernozem (G-HC), in Haplic Luvisol (G-HL) and in Eutric Vertisol (G-EV).

<p>Arrows indicate dominant fungal populations identified by sequencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044685#pone-0044685-t001" target="_blank">Table 1</a>). Band a: <i>Candida sake</i>; band c: <i>Fusarium</i> sp.; band d: <i>Gibberella zeae</i>; band e: <i>Verticillium dahliae</i>.</p