Background: Plants actively shape their associated microbial communities by synthesizing bio-active substances. Plant secondary metabolites are known for their signaling and plant defense functions, yet little is known about their overall effect on the plant microbiome. In this work, we studied the effects of benzoxazinoids (BXs), a group of secondary metabolites present in maize, on the host-associated microbial structure. Using BX knockout mutants and their W22 parental lines, we employed 16S and ITS2 rRNA gene amplicon analysis to characterize the maize microbiome at early growth stages. Results: Rhizo-box experiment showed that BXs affected microbial communities not only in roots and shoots, but also in the rhizosphere. Fungal richness in roots was more affected by BXs than root bacterial richness. Maize genotype (BX mutants and their parental lines) as well as plant age explained both fungal and bacterial community structure. Genotypic effect on microbial communities was stronger in roots than in rhizosphere. Diverse, but specific, microbial taxa were affected by BX in both roots and shoots, for instance, many plant pathogens were negatively correlated to BX content. In addition, a co-occurrence analysis of the root microbiome revealed that BXs affected specific groups of the microbiome. Conclusions: This study provides insights into the role of BXs for microbial community assembly in the rhizosphere and in roots and shoots. Coupling the quantification of BX metabolites with bacterial and fungal communities, we were able to suggest a gatekeeper role of BX by showing its correlation with specific microbial taxa and thus providing insights into effects on specific fungal and bacterial taxa in maize roots and shoots. Root microbial cooccurrence networks revealed that BXs affect specific microbial clusters.

Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches' Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches' broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host. . Phytoplasmas are obligate parasites that survive and replicate intracellularly within both insect and plant hosts. Within a plant, phytoplasmas inhabit the phloem and are injected directly into the cytoplasm of phloem sieve cells via the feeding activity of an insect vector. Insects capable of transmitting phytoplasmas comprise members of the order Hemiptera, including sap-sucking leafhoppers, planthoppers, and psyllids.Phytoplasmas have a plant host range that is in part dependent upon the feeding range of their insect vectors . Aster Yellows phytoplasma strain Witches' Broom (AY-WB; Candidatus Phytoplasma asteris) is vectored by the polyphagous aster leafhopper Macrosteles quadrilineatus, which readily transmits this pathogen to a wide range of plants, including members of the Solanaceae and Brassicaceae families (Sugio et al., 2011). Phytoplasmas such as AY-WB induce a variety of symptoms in plants that are indicative of an abnormal development of host tissues, including the formation of witches' broom (a dense mass of shoots originating from a single point), phyllody (conversion of floral organs into leaves), virescence (green pigmentation of tissues such as flower petals), and bolting (growth of elongated stalks). We hypothesized that phytoplasmas secrete virulence pro-

Phytoplasmas are cell wall-less bacteria inhabiting the phloem and utilizing it for their spread. Infected plants often show changes in growth pattern and a reduced crop yield. A quantitative real-time polymerase chain reaction (Q-PCR) assay and a bioimaging method were developed to quantify and localize phytoplasmas in situ. According to the Q-PCR assay, phytoplasmas accumulated disproportionately in source leaves of Euphorbia pulcherrima and, to a lesser extent, in petioles of source leaves and in stems. However, phytoplasma accumulation was small or nondetectable in sink organs (roots and sink leaves). For bioimaging, infected plant tissue was stained with vital fluorescence dyes and examined using confocal laser scanning microscopy. With a DNA-sensitive dye, the pathogens were detected exclusively in the phloem, where they formed dense masses in sieve tubes of Catharanthus roseus. Sieve tubes were identified by counterstaining with aniline blue for callose and multiphoton excitation. With a potentiometric dye, not all DNA-positive material was stained, suggesting that the dye stained metabolically active phytoplasmas only. Some highly infected sieve tubes contained phytoplasmas that were either inactive or dead upon staining.

In this paper, the recent progress within biosensors for plant pathogen detection will be reviewed. Bio-recognition layers on sensors can be designed in various ways, however the most popular approach is to immobilise antibodies for specific capture of analytes. Focus will be put on antibody surface-immobilisation strategies as well as the use of antibodies in the widely used sensors, quartz crystal microbalance, surface plasmon resonance and cantilevers. We will describe the available data on antibody-based plant pathogen detection and furthermore use examples from detection of the pathogens Salmonella, Listeria monocytogenes, Streptococcus mutans, Bacillus cereus, Bacillus anthracis, Campylobacter and Escherichia coli. We will touch upon optimal assay design and further discuss the strengths and limitations of current sensor technologies for detection of viruses, bacteria and fungi.

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SummaryVirus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for studies of gene function. However, efficient VIGS has only been accomplished in a few plant species. In order to extend the application of VIGS, we examined whether a VIGS vector based on Pea early browning virus (PEBV) would produce recognizable phenotypes in Pisum sativum. A plasmid vector of PEBV was modified to allow agro-inoculation and insertion of heterologous sequences. cDNA fragments of the P. sativum phytoene desaturase (PDS), LEAFY (LFY) and KORRIGAN1 (KOR1) homologues were inserted into the PEBV RNA2 vector, replacing the genes required for nematode transmission. Pisum sativum inoculated with PEBV carrying a fragment of PsPDS developed characteristic photo-bleached leaves and this phenotype was associated with a significant reduction in PsPDS mRNA. The P. sativum homologue of LFY is known as UNIFOLIATA (UNI). Plants inoculated with PEBV carrying a fragment of UNI developed distorted flowers and leaves with modified architecture, which are also observed in UNI-mutants. In Arabidopsis thaliana, the KOR1-mutant is characterized by an extreme dwarf phenotype. Pisum sativum plants inoculated with PEBV carrying a fragment of PsKOR1 displayed a significant reduction in height and inhibition of root growth. The PEBV VIGS vector did not affect the ability of P. sativum to flower, set seeds, and form nodules characteristic of symbiosis with rhizobium. These results suggest that the PEBV vector can be applied to functional genomics in a legume species to study genes involved in a wide range of biological processes.

SummaryThe phyllosphere mycobiome in cereals is an important determinant of crop health. However, an understanding of the factors shaping this community is lacking.Fungal diversity in leaves from a range of cultivars of winter wheat (Triticum aestivum), winter and spring barley (Hordeum vulgare) and a smaller number of samples from oat (Avena sativa), rye (Secale cereale) and triticale (Triticum 9 Secale) was studied using next-generation sequencing. The effects of host genotype, fungicide treatment and location on fungal communities were explored.In total, 635 251 fungal internal transcribed spacer (ITS) reads were obtained from 210 leaf samples. Visual disease assessments and relative read abundance of Zymoseptoria tritici and Ramularia collo-cygni were strongly positively related. Crop genotype at the species level explained 43% of the variance in the total dataset, followed by fungicide treatment (13%) and location (4%). Indicator species, including plant pathogens, responding to factors such as crop species, location and treatment were identified.Host genotype at both the species and cultivar level is important in shaping phyllosphere fungal communities, whereas fungicide treatment and location have minor effects. We found many host-specific fungal pathogens, but also a large diversity of fungi that were relatively insensitive to host genetic background, indicating that host-specific pathogens live in a 'sea' of nonspecific fungi.

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