Bioengineering

Recirculating aquaculture systems (RAS) are promising candidates for the sustainable development of the aquaculture industry. A current limitation of RAS is the production and potential accumulation of nitrogenous wastes, ammonium (NH4+), nitrite (NO2−) and nitrate (NO3−), which could affect fish health and welfare. In a previous experiment, we have demonstrated that the marine anammox bacteria Candidatus Scalindua was a promising candidate to treat the wastewater (WW) of marine, cold-water RAS. However, the activity of the bacteria was negatively impacted after a direct exposure to RAS WW. In the current study, we have further investigated the potential of Ca. Scalindua to treat marine RAS WW in a three-phase experiment. In the first phase (control, 83 days), Ca. Scalindua was fed a synthetic feed, enriched in NH4+, NO2− and trace element (TE) mix. Removal rates of 98.9% and 99.6% for NH4+ and NO2−, respectively, were achieved. In the second phase (116 days), we gradually increased the exposure of Ca. Scalindua to nitrogen-enriched RAS WW over a period of about 80 days. In the last phase (79 days), we investigated the needs of TE supplementation for the Ca. Scalindua after they were fully acclimated to 100% RAS WW. Our results show that the gradual exposure of Ca. Scalindua resulted in a successful acclimation to 100% RAS WW, with maintained high removal rates of both NH4+ and NO2− throughout the experiment. Despite a slight decrease in relative abundance (from 21.4% to 16.7%), Ca. Scalindua remained the dominant species in the granules throughout the whole experiment. We conclude that Ca. Scalindua can be successfully used to treat marine RAS WW, without the addition of TE, once given enough time to acclimate to its new substrate. Future studies need to determine the specific needs for optimal RAS WW treatment by Ca. Scalindua at pilot scale.

Summary Huanglongbing (HLB) is currently considered the most destructive disease of citrus worldwide. In the major citrus‐growing areas in Asia and the US, the major causal agent of HLB is the bacterial pathogen Candidatus Liberibacter asiaticus (CLas). CLas is vectored by the Asian citrus psyllid, Diaphorina citri , in a persistent propagative manner. CLas cannot be cultured in vitro because of its unclear growth factors, leading to uncertainty in the infection mechanism of CLas at the cellular level in citrus and in D. citri . To characterize the detailed infection of CLas in the host and vector, the incidence of HLB was first investigated in citrus‐growing fields in Fujian Province, China. It was found that the positive association of the level of CLas infection in the leaves correlated with the symptoms. Then antibodies against peptides of the outer membrane protein (OMP) of CLas were prepared and tested. The antibodies OMP‐225, OMP‐333 and OMP724 showed specificity to citrus plants in western blot analyses, whereas the antibodies OMP‐47 and OMP‐225 displayed specificity to the D. citri vector. The application of OMP‐225 in the immunofluorescence assay indicated that CLas was located in and distributed throughout the phloem sieve cells of the leaf midribs and axile placenta of the fruit. CLas also infected the epithelial cells and visceral muscles of the alimentary canal of D. citri . The application of OMP‐333 in immunoelectron microscopy indicated the round or oval CLas in the sieve cells of leaf midribs and axile placenta of fruit as well as in the epithelial cells and reticular tissue of D. citri alimentary canal. These results provide a reliable means for HLB detection, and enlighten a strategy via neutralizing OMP to control HLB. These findings also provide insight for the further investigation on CLas infection and pathogenesis, as well as CLas–vector interaction.

Summary ‘ Candidatus Liberibacter asiaticus’ (CLas) is a phloem‐limited non‐culturable α‐proteobacterium associated with citrus Huanglongbing, a highly destructive disease threatening global citrus industry. Research on CLas is challenging due to the current inability to culture CLas in vitro and the low CLas titre in citrus plant. Here, we develop a CLas enrichment system using the holoparasitic dodder plant ( Cuscuta campestris ) as an amenable host to acquire and enrich CLas from CLas‐infected citrus shoots maintained hydroponically. Forty‐eight out of fifty‐five (87%) dodder plants successfully parasitized CLas‐infected citrus shoots with detectable CLas by PCR. Among 48 dodders cultures, 30 showed two‐ to 419‐fold CLas titre increase as compared to the corresponding citrus hosts. The CLas population rapidly increased and reached the highest level in dodder tendrils at 15 days after parasitizing citrus shoot. Genome sequencing and assembly derived from CLas‐enriched dodder DNA samples generated a higher resolution than those obtained for CLas from citrus hosts. No genomic variation was detected in CLas after transmission from citrus to dodder during short‐term parasitism. Dual RNA‐Seq experiments showed similar CLas gene expression profiles in dodder and citrus samples, yet dodder samples generated a higher resolution of CLas transcriptome data. The ability of dodder to support CLas multiplication to high levels, as well as its advantage in CLas genomic and transcriptomic analyses, make it an optimal model for further studies on CLas–host interaction.

Summary ‘ Candidatus Liberibacter asiaticus ’ is a fastidious bacterium and a putative agent of citrus greening disease (a.k.a., huanglongbing, HLB), a significant agricultural disease that affects citrus fruit quality and tree health. In citrus, ‘ Ca. L. asiaticus ’ is phloem limited. Lack of culture tools to study ‘ Ca. L. asiaticus ’ complicates analysis of this important organism. To improve understanding of ‘ Ca. L. asiaticus ’–host interactions including parameters that affect ‘ Ca. L. asiaticus ’ replication, methods suitable for screening pathogen responses to physicochemical and nutritional variables are needed. We describe a leaf disc‐based culture assay that allows highly selective measurement of changes in ‘ Ca. L. asiaticus ’ DNA within plant tissue incubated under specific physicochemical and nutritional conditions. qPCR analysis targeting the hypothetical gene CD16‐00155 (strain A4) allowed selective quantification of ‘ Ca. L. asiaticus ’ DNA content within infected tissue. ‘ Ca. L. asiaticus ’ DNA replication was observed in response to glucose exclusively under microaerobic conditions, and the antibiotic amikacin further enhanced ‘ Ca. L. asiaticus ’ DNA replication. Metabolite profiling revealed a moderate impact of ‘ Ca. L. asiaticus ’ on the ability of leaf tissue to metabolize and respond to glucose.