The Microbial Diversity of Cabbage Pest Delia radicum Across Multiple Life Stages.

The cabbage root fly Delia radicum is a worldwide pest that causes yield losses of many common cabbage crops. The bacteria associated with D. radicum are suggested to influence the pest status of their host. In this study, we characterized insect-associated bacteria of D. radicum across multiple life stages and of their diet plant (turnip, Brassica rapa subsp. rapa) by sequencing the V3-V4 region of 16S rRNA genes using the Illumina MiSeq platform. In total, over 1.2M paired-end reads were obtained, identifying 1006 bacterial amplicon sequence variants (ASVs) in samples obtained from the eggs, larvae, pupae and adults of D. radicum, as well as turnips that were either fresh or infested with D. radicum larvae. The microbial community in D. radicum was dominated by Wolbachia, a common endosymbiont of arthropods which we found in all of the investigated insect samples, with the pupal stage having the highest relative abundance. Moderate amounts of Firmicutes were found only in adult D. radicum flies, but not in previous life stages. Actinobacteria were mostly found on the eggs and on the skin of fresh plants on which the eggs were deposited. These plants also harbored a large amount of Pseudomonas. The bacterial diversity of the healthy turnip was low, whereas the microbial community of decaying turnips that were heavily infested by D. radicum larvae and showing symptoms of advanced soft rot was characterized by a high bacterial diversity. Taken together, this work provides insights into the bacterial communities associated with the cabbage pest D. radicum and its associated disease symptoms.

Single gene enables plant pathogenic Pectobacterium to overcome host-specific chemical defence.

Plants of the Brassicales order, including Arabidopsis and many common vegetables, produce toxic isothiocyanates to defend themselves against pathogens. Despite this defence, plant pathogenic microorganisms like Pectobacterium cause large yield losses in fields and during storage of crops. The bacterial gene saxA was previously found to encode isothiocyanate hydrolase that degrades isothiocyanates in vitro. Here we demonstrate in planta that saxA is a virulence factor that can overcome the chemical defence system of Brassicales plants. Analysis of the distribution of saxA genes in Pectobacterium suggests that saxA from three different phylogenetic origins are present within this genus. Deletion of saxA genes representing two of the most common classes from P. odoriferum and P. versatile resulted in significantly reduced virulence on Arabidopsis thaliana and Brassica oleracea. Furthermore, expressing saxA from a plasmid in a potato-specific P. parmentieri strain that does not naturally harbour this gene significantly increased the ability of the strain to macerate Arabidopsis. These findings suggest that a single gene may have a significant role in defining the host range of a plant pathogen.

Co-cultivation of the strictly anaerobic methanogen Methanosarcina barkeri with aerobic methanotrophs in an oxygen-limited membrane bioreactor.

Wetlands contribute to 30% of global methane emissions due to an imbalance between microbial methane production and consumption. Methanogenesis and methanotrophy have mainly been studied separately, and little is known about their potential interactions in aquatic environments. To mimic the interaction between methane producers and oxidizers in the environment, we co-cultivated the methanogenic archaeon Methanosarcina barkeri with aerobic Methylocystaceae methanotrophs in an oxygen-limited bioreactor using acetate as methanogenic substrate. Methane, acetate, dissolved oxygen, available nitrogen, pH, temperature, and cell density were monitored to follow system stability and activity. Stable reactor operation was achieved for two consecutive periods of 2 months. Fluorescence in situ hybridization micrographs indicated close association between both groups of microorganisms. This association suggests that the methanotrophs profit from direct access to the methane that is produced from acetate, while methanogens are protected by the concomitant oxygen consumption of the methanotrophs. This proof of principle study can be used to set up systems to study their responses to environmental changes.

Functional Profiling and Crystal Structures of Isothiocyanate Hydrolases Found in Gut-Associated and Plant-Pathogenic Bacteria.

Isothiocyanates (ITCs) are produced by cruciferous plants to protect them against herbivores and infection by microbes. These compounds are of particular interest due to their antimicrobial and anticarcinogenic properties. The breakdown of ITCs in nature is catalyzed by isothiocyanate hydrolases (ITCases), a novel family within the metallo-ß-lactamase (MBL)-fold superfamily of proteins. saxA genes that code for ITCases are particularly widespread in insect- and plant-associated bacteria. Enzymatic characterization of seven phylogenetically related but distinct ITCases revealed similar activities on six selected ITCs, suggesting that phylogenetic diversity does not determine the substrate specificity of ITCases. X-ray crystallography studies of two ITCases sharing 42% amino acid sequence identity revealed a highly conserved tertiary structure. Notable features of ITCases include a hydrophobic active site with two Zn2+ ions coordinating water/hydroxide and a flexible cap that is implicated in substrate recognition and covers the active site. This report reveals the function and structure of the previously uncharacterized family of isothiocyanate hydrolases within the otherwise relatively well-studied superfamily of metallo-ß-lactamases.IMPORTANCE This study explores a newly discovered protein in the ß-lactamase superfamily, namely, SaxA, or isothiocyanate hydrolase. Isothiocyanates are defensive compounds found in many cabbage-related crop plants and are currently being investigated for their antimicrobial and anticarcinogenic properties. We show that isothiocyanate hydrolases are responsible for the breakdown of several of these plant defensive chemicals in vitro and suggest their potential for mitigating the beneficial effects of isothiocyanates in crop protection and cancer prevention.

Detoxifying symbionts in agriculturally important pest insects.

Pest insects lead to excessive agricultural and therefore economical losses on crops worldwide. These insects have to withstand toxic molecules that are inherent to plant defences, as well as those that are produced and introduced by humans in the form of insecticides. In recent years, research on insect-microbe symbioses has recognized that microbial symbionts may play a role protecting against these toxins, leading to a form of defensive symbiosis between the pest insect and different types of microorganisms that we term detoxifying symbioses. In this minireview, we will highlight well-characterized and emerging insect model systems of detoxifying symbioses and assess how the microorganisms influence the host's success.

Plasmids from the gut microbiome of cabbage root fly larvae encode SaxA that catalyses the conversion of the plant toxin 2-phenylethyl isothiocyanate.

Cabbage root fly larvae (Delia radicum) cause severe crop losses (≥ 50%) of rapeseed/ canola and cabbages used in the food and biofuel industries. These losses occur despite the fact that cabbages produce insecticidal toxins such as isothiocyanates. Here we describe the cabbage root fly larval gut microbiome as a source of isothiocyanate degrading enzymes. We sequenced the microbial gut community of the larvae and analysed phylogenetic markers and functional genes. We combined this with the isolation of several microbial strains representing the phylogenetic distribution of the metagenome. Eleven of those isolates were highly resistant towards 2-phenylethyl isothiocyanate, a subset also metabolized 2-phenylethyl isothiocyanate. Several plasmids appeared to be shared between those isolates that metabolized the toxin. One of the plasmids harboured a saxA gene that upon transformation gave resistance and enabled the degradation of 2-phenylethyl isothiocyanate in Escherichia coli. Taken together, the results showed that the cabbage root fly larval gut microbiome is capable of isothiocyanate degradation, a characteristic that has not been observed before, and may help us understand and design new pest control strategies.