Joana Beatrice Meyer

Interplay between Wheat Cultivars, Biocontrol Pseudomonads, and Soil

ABSTRACT There is a significant potential to improve the plant-beneficial effects of root-colonizing pseudomonads by breeding wheat genotypes with a greater capacity to sustain interactions with these bacteria. However, the interaction between pseudomonads and crop plants at the cultivar level, as well as the conditions which favor the accumulation of beneficial microorganisms in the wheat rhizosphere, is largely unknown. Therefore, we characterized the three Swiss winter wheat (Triticum aestivum) cultivars Arina, Zinal, and Cimetta for their ability to accumulate naturally occurring plant-beneficial pseudomonads in the rhizosphere. Cultivar performance was measured also by the ability to select for specific genotypes of 2,4-diacetylphloroglucinol (DAPG) producers in two different soils. Cultivar-specific differences were found; however, these were strongly influenced by the soil type. Denaturing gradient gel electrophoresis (DGGE) analysis of fragments of the DAPG biosynthetic gene phlD amplified from natural Pseudomonas rhizosphere populations revealed that phlD diversity substantially varied between the two soils and that there was a cultivar-specific accumulation of certain phlD genotypes in one soil but not in the other. Furthermore, the three cultivars were tested for their ability to benefit from Pseudomonas inoculants. Interestingly, Arina, which was best protected against Pythium ultimum infection by inoculation with Pseudomonas fluorescens biocontrol strain CHA0, was the cultivar which profited the least from the bacterial inoculant in terms of plant growth promotion in the absence of the pathogen. Knowledge gained of the interactions between wheat cultivars, beneficial pseudomonads, and soil types allows us to optimize cultivar-soil combinations for the promotion of growth through beneficial pseudomonads. Additionally, this information can be implemented by breeders into a new and unique breeding strategy for low-input and organic conditions.

Pyrroloquinoline quinone biosynthesis gene pqqC, a novel molecular marker for studying the phylogeny and diversity of phosphate-solubilizing pseudomonads.

ABSTRACT Many root-colonizing pseudomonads are able to promote plant growth by increasing phosphate availability in soil through solubilization of poorly soluble rock phosphates. The major mechanism of phosphate solubilization by pseudomonads is the secretion of gluconic acid, which requires the enzyme glucose dehydrogenase and its cofactor pyrroloquinoline quinone (PQQ). The main aim of this study was to evaluate whether a PQQ biosynthetic gene is suitable to study the phylogeny of phosphate-solubilizing pseudomonads. To this end, two new primers, which specifically amplify the pqqC gene of the Pseudomonas genus, were designed. pqqC fragments were amplified and sequenced from a Pseudomonas strain collection and from a natural wheat rhizosphere population using cultivation-dependent and cultivation-independent approaches. Phylogenetic trees based on pqqC sequences were compared to trees obtained with the two concatenated housekeeping genes rpoD and gyrB. For both pqqC and rpoD-gyrB, similar main phylogenetic clusters were found. However, in the pqqC but not in the rpoD-gyrB tree, the group of fluorescent pseudomonads producing the antifungal compounds 2,4-diacetylphloroglucinol and pyoluteorin was located outside the Pseudomonas fluorescens group. pqqC sequences from isolated pseudomonads were differently distributed among the identified phylogenetic groups than pqqC sequences derived from the cultivation-independent approach. Comparing pqqC phylogeny and phosphate solubilization activity, we identified one phylogenetic group with high solubilization activity. In summary, we demonstrate that the gene pqqC is a novel molecular marker that can be used complementary to housekeeping genes for studying the diversity and evolution of plant-beneficial pseudomonads.

Does Wheat Genetically Modified for Disease Resistance Affect Root-Colonizing Pseudomonads and Arbuscular Mycorrhizal Fungi?

This study aimed to evaluate the impact of genetically modified (GM) wheat with introduced pm3b mildew resistance transgene, on two types of root-colonizing microorganisms, namely pseudomonads and arbuscular mycorrhizal fungi (AMF). Our investigations were carried out in field trials over three field seasons and at two locations. Serial dilution in selective Kings B medium and microscopy were used to assess the abundance of cultivable pseudomonads and AMF, respectively. We developed a denaturing gradient gel electrophoresis (DGGE) method to characterize the diversity of the pqqC gene, which is involved in Pseudomonas phosphate solubilization. A major result was that in the first field season Pseudomonas abundances and diversity on roots of GM pm3b lines, but also on non-GM sister lines were different from those of the parental lines and conventional wheat cultivars. This indicates a strong effect of the procedures by which these plants were created, as GM and sister lines were generated via tissue cultures and propagated in the greenhouse. Moreover, Pseudomonas population sizes and DGGE profiles varied considerably between individual GM lines with different genomic locations of the pm3b transgene. At individual time points, differences in Pseudomonas and AMF accumulation between GM and control lines were detected, but they were not consistent and much less pronounced than differences detected between young and old plants, different conventional wheat cultivars or at different locations and field seasons. Thus, we conclude that impacts of GM wheat on plant-beneficial root-colonizing microorganisms are minor and not of ecological importance. The cultivation-independent pqqC-DGGE approach proved to be a useful tool for monitoring the dynamics of Pseudomonas populations in a wheat field and even sensitive enough for detecting population responses to altered plant physiology.

Interaction between two invasive organisms on the European chestnut: does the chestnut blight fungus benefit from the presence of the gall wasp?

The impact of invasive fungal pathogens and pests on trees is often studied individually, thereby omitting possible interactions. In this study the ecological interaction between the chestnut blight fungus Cryphonectria parasitica and the chestnut gall wasp Dryocosmus kuriphilus was investigated. We determined if abandoned galls could be colonized by C. parasitica and thereby act as an entry point and a source of pathogen inoculum. Moreover we assessed the identity and diversity of other gall-colonizing fungal species. A total of 1973 galls were randomly sampled from 200 chestnut trees in eight Swiss stands. In a stand C. parasitica was isolated from 0.4-19.2% of the galls. The incidence of C. parasitica on the galls and the fungal diversity significantly increased with the residence time of D. kuriphilus in a stand. All but one C. parasitica cultures were virulent. The predominant fungus isolated from galls was Gnomoniopsis castanea whose abundance influenced negatively that of C. parasitica. This study shows that D. kuriphilus galls can be colonized by virulent strains of the chestnut blight fungus C. parasitica. This can have effects on the chestnut blight incidence even in chestnut stands where the disease is successfully controlled by hypovirulence. The gall wasp presence influences also the fungal species composition on chestnut trees.

Novel T-RFLP method to investigate six main groups of 2,4-diacetylphloroglucinol-producing pseudomonads in environmental samples

Strains of fluorescent pseudomonads producing 2,4-diacetylphloroglucinol (DAPG) are involved in the protection of plant roots against soil-borne plant pathogens. Recently, a multilocus sequence analysis of a world wide collection of DAPG-producers led to the identification of six main groups (A-F). In this study a T-RFLP method based on the phlD gene was developed to efficiently identify the members of these six groups in environmental samples. A combination of six restriction enzymes was identified which leads to group specific terminal fragments (T-RF). The detection limit of the phlD-T-RFLP method was determined for the two P. fluorescens strains F113 (group B) and CHA0 (group F) in rhizosphere samples and was found to be 5×10(3)CFU/g and 5×10(4)CFU/g respectively. PhlD-T-RFLP and phlD-DGGE analysis of wheat and maize root samples from greenhouse and field revealed similarly the presence of multilocus groups A, B and D. However, they were more frequently detected with phlD-T-RFLP. Additionally, groups C and F were detected in greenhouse samples but only by phlD-T-RFLP and not by phlD-DGGE. In conclusion, the new phlD-T-RFLP method proved to be a fast and reliable method to detect strains of the six main groups of DAPG-producers in environmental samples with an improved detection limit compared to phlD-DGGE.

DNA fingerprinting of pearls to determine their origins.

We report the first successful extraction of oyster DNA from a pearl and use it to identify the source oyster species for the three major pearl-producing oyster species Pinctada margaritifera, P. maxima and P. radiata. Both mitochondrial and nuclear gene fragments could be PCR-amplified and sequenced. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay in the internal transcribed spacer (ITS) region was developed and used to identify 18 pearls of unknown origin. A micro-drilling technique was developed to obtain small amounts of DNA while maintaining the commercial value of the pearls. This DNA fingerprinting method could be used to document the source of historic pearls and will provide more transparency for traders and consumers within the pearl industry.

Phylogenetic and phenotypic characterisation of Sirococcus castaneae comb. nov (synonym Diplodina castaneae), a fungal endophyte of European chestnut

In this paper we resolve the taxonomic status of the fungus Diplodina castaneae (Ascomycetes, Diaporthales, Gnomoniaceae) which occurs on the European chestnut (Castanea sativa) as endophyte and as the causal agent of Javart disease. Specimens from Switzerland, Spain, and Azerbaijan were sequenced at five nuclear loci (β-tubulin, EF-1α, ITS, LSU, and RPB2). Phylogenies were inferred to place D. castaneae in the Gnomoniaceae family. Moreover, growth rates and morphological characteristics on different agar media were assessed and compared to those of Gnomoniopsis castaneae, which can easily be confused with D. castaneae. Based on morphological and phylogenetic characteristics, we propose to reallocate D. castaneae to the genus Sirococcus, as S. castaneae comb. nov.