Publications

4483

AbstractAnaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and mixotrophy beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus ‘Kuenenia stuttgartiensis’ using time-series 13C and 2H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis (INST-MFA). Our findings confirm predicted metabolic pathways used for CO2 fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis, and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of an oxidative tricarboxylic acid cycle, despite the genome not encoding a known citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood-Ljungdahl pathway instead of oxidizing it completely to CO2 followed by reassimilation. In contrast, our data suggests that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor’s side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.

AbstractBois noir (BN), the most prevalent disease of the grapevine yellows complex, causes considerable yield loss in vineyards. BN is associated with phytoplasma strains of the species ‘Candidatus Phytoplasma solani’ (taxonomic subgroup 16SrXII‐A). In Europe, the BN phytoplasma is transmitted to grapevine mainly by Hyalesthes obsoletus, a polyphagous cixiid completing its life cycle on stinging nettle and field bindweed. As a result of the complexity of BN epidemiology, no effective control strategies have been developed. In previous studies conducted in the eastern Mediterranean coast of Israel, chaste tree (Vitex agnus‐castus) was found to be the preferred host plant of H. obsoletus but did not harbour BN phytoplasma. Thus, a ‘push and pull’ strategy was suggested based on the fact that chaste tree plants located at vineyard borders was an effective trap plant for H. obsoletus adults. However, in other studies carried out in the eastern Adriatic coast of Montenegro, chaste tree was found to be a key source plant for BN phytoplasma transmission to grapevine. This study aimed to investigate (i) the interaction between chaste tree and H. obsoletus through survival, attractiveness and oviposition experiments conducted comparing the behaviour of H. obsoletus in chaste tree versus stinging nettle and grapevine and (ii) the capability of chaste tree to harbor ‘Ca. P. solani’ in northern Italy through transmission trials. H. obsoletus adults were found to survive on chaste tree and grapevine over a 1 week period and prefer chaste tree to grapevine. Moreover, H. obsoletus produced eggs and overwintered as nymphs on chaste tree, even if at a lesser extent than on stinging nettle. H. obsoletus originating from nettle was found able to transmit ‘Ca. P. solani’ to chaste tree (2 plants of 16 were found infected by the BN phytoplasma strain St5 identified in H. obsoletus specimens). These results increased our knowledge about the role of Vitex agnus‐castus as host plant of H. obsoletus and BN phytoplasma in northern Italy and do not recommend considering chaste tree as trap plant at vineyard borders.

Phytoplasmas are the causative agent of numerous diseases of plant species all over the world, including important food crops. The mode by which phytoplasmas multiply and behave in their host is poorly understood and often based on genomic data. We used yeast two-hybrid screening to find new protein–protein interactions between the causal agent of apple proliferation ‘Candidatus Phytoplasma mali’ and its host plant. Here, we report that the ‘Ca. P. mali’ strain PM19 genome encodes a protein PM19_00185 that interacts with at least six different ubiquitin-conjugating enzymes (UBC; E2) of Arabidopsis thaliana. An in vitro ubiquitination assay showed that PM19_00185 is enzymatically active as E3 ligase with A. thaliana E2 UBC09 and Malus domestica E2 UBC10. We show that a nonhost bacteria (Pseudomonas syringae pv. tabaci) can grow in transgenic A. thaliana plant lines expressing PM19_00185. A connection of phytoplasma effector proteins with the proteasome proteolytic pathway has been reported before. However, this is, to our knowledge, the first time that a phytoplasma effector protein with E3 ligase activity has been reported.

Liberibacter crescens is a bacterium that is closely related to plant pathogens that have caused billions of dollars in crop losses in recent years. Particularly devastating are citrus losses due to citrus greening disease, also known as Huanglongbing, which is caused by “ Candidatus Liberibacter asiaticus” and carried by the Asian citrus psyllid. L. crescens is the only close relative of “ Ca . Liberibacter asiaticus” that can currently be grown in culture, and it therefore serves as an important model organism for the growth, genetic manipulation, and biological control of the pathogenic species. Here, we show that one of the greatest limitations to L. crescens growth is the sharp increase in alkaline conditions it produces as a consequence of consumption of its preferred nutrient source. In addition to new information about L. crescens growth and metabolism, we provide new guidelines for culture conditions that improve the survival and yield of L. crescens .

AbstractThe epidemiology of ‘Candidatus Phytoplasma prunorum’ was studied in Austria and Hungary from 2014 to 2018. Testing of root samples showed average infections rates of 61 and 40% of the Austrian Prunus spinosa and Prunus domestica spp. insititia samples, respectively. In Hungary, on average 21% of the P. spinosa and 13% of the feral Prunus cerasifera samples were infected. The pathogen was found in 18 out of 19 apricot orchards and PCR positive Cacopsylla pruni were observed at 11 out of 17 sampling locations in both countries. In cage experiments with C. pruni remigrants successful pathogen transmission to Prunus armeniaca, P. domestica and P. spinosa seedlings in budding and foliated developmental stages was recorded, an inoculation access period of 4 hr was sufficient for transmission. A field experiment with ungrafted apricot seedlings planted in 2012 and 2014 indicated a prominent role of the insect vectors for disease spread. In 2017, 40 and 28% of the trees planted in 2012 and 2014, respectively, were infected. Molecular characterisation based on the genes aceF and imp allowed the discrimination between 10 phytoplasma types in apricots. Around 70% of the phytoplasma types in apricots were also common in P. spinosa, in P. domestica spp. insititia and in remigrant C. pruni pointing to a possible pathogen exchange by insects between wild and cultivated Prunus spp. For disease control, vector management over the entire flight period of the remigrants seems necessary; when selecting active compounds, the short inoculation access period of not more than 4 hr should be considered.