Publications

4505

Abstract Anaerobic ammonium oxidation (Anammox) bacteria are a group of extraordinary bacteria exerting a major impact on the global nitrogen cycle. Their phylogenetic breadth and diversity, however, are not well constrained. Here we describe a new, deep-branching family in the order of Candidatus Brocadiales, Candidatus Bathyanammoxibiaceae, members of which have genes encoding the key enzymes of the anammox metabolism. In marine sediment cores from the Arctic Mid-Ocean Ridge (AMOR), the presence of Ca. Bathyanammoxibiaceae was confined within the nitrate-ammonium transition zones with the counter gradients of nitrate and ammonium, coinciding with the predicted occurrence of the anammox process. Ca. Bathyanammoxibiaceae genomes encode the core genetic machinery for the anammox metabolism, including hydrazine synthase for converting nitric oxide and ammonium to hydrazine, and hydrazine dehydrogenase for hydrazine oxidation to dinitrogen gas, and hydroxylamine oxidoreductase for nitrite reduction to nitric oxide. Their occurrences assessed by genomes and 16S rRNA gene sequencings surveys indicate that they are present in both marine and terrestrial environments. By introducing the anammox potential of Ca. Bathyanammoxibiaceae and charactering their ideal niche in marine sediments, our findings suggest that the diversity and abundance of anammox bacteria may be higher than previously thought, and provide important insights on cultivating them in the future to not only assess their biogeochemical impacts but also constrain the emergence and evolutionary history of this functional guild on Earth.

Abstract Objective ‘Candidatus Liberibacter asiaticus’ (CLas) is associated with the devastating citrus ‘greening’ disease. All attempts to achieve axenic growth and complete Koch’s postulates with CLas have failed to date, at best yielding complex cocultures with very low CLas titers detectable only by PCR. Reductive genome evolution has rendered all pathogenic ‘Ca. Liberibacter’ spp. deficient in multiple key biosynthetic, metabolic and structural pathways that are highly unlikely to be rescued in vitro by media supplementation alone. By contrast, Liberibacter crescens (Lcr) is axenically cultured and its genome is both syntenic and highly similar to CLas. Our objective is to achieve replicative axenic growth of CLas via addition of missing culturability-related Lcr genes. Results Bioinformatic analyses identified 405 unique ORFs in Lcr but missing (or truncated) in all 24 sequenced CLas strains. Site-directed mutagenesis confirmed and extended published EZ-Tn5 mutagenesis data, allowing elimination of 310 of these 405 genes as nonessential, leaving 95 experimentally validated Lcr genes as essential for CLas growth in axenic culture. Experimental conditions for conjugation of large GFP-expressing plasmids from Escherichia coli to Lcr were successfully established for the first time, providing a practical method for transfer of large groups of ‘essential’ Lcr genes to CLas.

Abstract Background The appearance of the novel porcine haemotrophic mycoplasma (HM) species ‘Candidatus Mycoplasma haemosuis’ was reported in apparently healthy but also in clinically sick animals in China, Korea and in a case report from Germany. Outside of Asia, however, nothing further is known about the frequency of ‘Ca. M. haemosuis’ in pigs to date. To investigate the distribution of this novel HM species in Germany, fattening pigs, sows and pre-suckling piglets were examined using a herein developed quantitative real-time PCR assay (qPCR). Because the piglets were sampled before the first colostrum uptake, additional information on a possible vertical transmission from dams to their offspring was obtained. Results Our novel qPCR assay successfully detected ‘Ca. M. haemosuis’ in all blood samples from the ‘Ca. M. haemosuis’-infected pigs. No cross-reactivity was detected when DNA from non-target Mycoplasma spp. and other bacterial species representing 105 bacteria/reaction were used as a template. The lower limit of detection of the qPCR was thus 10 gap gene copies per reaction and 2.5 x 103 genome equivalents (GE) per mL blood. ‘Candidatus M. haemosuis’ was detected by this qPCR in blood samples from a total out of 6.25% sows (13/208), 4.50% pre-suckling piglets (28/622) and 17.50% fattening pigs (35/200). On farm level, 3 out of 21 piglet producing farms (14.28%) and 9 out of 20 fattening farms (45.00%) were positive for ‘Ca. M. haemosuis’. Co-infections with M. suis were evident in all age groups. Conclusion ‘Candidatus M. haemosuis’ infection is present in German pig farms and the detection of the novel porcine HM species in piglets immediately after birth before colostrum intake indicates vertical transmission. The novel qPCR assay specific for ‘Ca. M. haemosuis’ described herein will be a prerequisite for future studies on the prevalence, epidemiology as well as the clinical and economic impact of ‘Ca. M. haemosuis’ infections.

AbstractBACKGROUNDAsian citrus psyllid (ACP), also known as Diaphorina citri, is the natural vector of Candidatus Liberibacter asiaticus (CLas), which is responsible for Huanglongbing (HLB), a devastating citrus disease. Previously, the pathogen was successfully excluded from diseased citrus plants by using the indigenous endophyte Bacillus subtilis L1‐21. However, the pathogen elimination and colonization potential of B. subtilis L1‐21 in the carrier vector ACP, as well as the recruitment of native microbial communities of psyllid in the presence of endophytes, are still unknown.RESULTSInitially, we suggested that endophyte L1‐21 reduced the CLas copies in ACP from 6.58 × 106 to 5.04 × 104 per insect after 48 h, however, the pathogen copies remained stable in the negative control. The endophyte was stable for 48 h after application. Among the bacterial genera those highlighted in ACP were Candidatus Liberibacter, Pseudomonas, Candidatus Profftella, Methylobacterium‐Methylorubrum, Pantoea, Curtobacterium, Wolbachia, Actinomycetospora, and Bacillus. Interestingly, B. subtilis L1‐21 easily colonizes the midgut of ACP but cannot be detected in eggs. When ACP with endophyte L1‐21 was allowed to feed on new citrus leaves, the highest colonization was observed. We also found that psyllids carrying endophyte L1‐21 after feeding on citrus leaves reduced the CLas copies in leaves on the 0, 3rd and 5th day from 8.18 × 10,4 2.6 × 10,3 and 0 pathogen copies/g fresh midvein, respectively.CONCLUSIONSWe propose that B. subtilis L1‐21 is a native endophyte in citrus and psyllid, which efficiently reduces the CLas pathogen in both citrus and psyllids, provides a more protective effect by increasing the number of cultivable endophytes, and successfully colonizes the midgut of ACP. © 2022 Society of Chemical Industry.

SUMMARY‘Candidatus Phytoplasma tritici’ (‘Ca. P. tritici’) is an insect‐borne obligate pathogen that infects wheat (Triticum aestivum) causing wheat blue dwarf disease, and leads to yield losses. SWP12 is a potential effector secreted by ‘Ca. P. tritici’ that manipulates host processes to create an environment conducive to phytoplasma colonization, but the detailed mechanism of action remains to be investigated. In this study, the expression of SWP12 weakened the basal immunity of Nicotiana benthamiana and promoted leaf colonization by Phytophthora parasitica, Sclerotinia sclerotiorum, and tobacco mild green mosaic virus. Moreover, the expression of SWP12 in wheat plants promoted phytoplasma colonization. Triticum aestivum WRKY74 and N. benthamiana WRKY17 were identified as host targets of SWP12. The expression of TaWRKY74 triggered reactive oxygen species bursts, upregulated defense‐related genes, and decreased TaCRR6 transcription, leading to reductions in NADH dehydrogenase complex (NDH) activity. Expression of TaWRKY74 in wheat increased plant resistance to ‘Ca. P. tritici’, and silencing of TaWRKY74 enhanced plant susceptibility, which indicates that TaWRKY74 is a positive regulator of wheat resistance to ‘Ca. P. tritici’. We showed that SWP12 weakens plant resistance and promotes ‘Ca. P. tritici’ colonization by destabilizing TaWRKY74.