Ecology, Evolution, Behavior and Systematics

ABSTRACT Filamentous Chloroflexota are abundant in activated sludge wastewater treatment plants (WWTPs) worldwide and are occasionally associated with poor solid-liquid separation or foaming, but most of the abundant lineages remain undescribed. Here, we present a comprehensive overview of Chloroflexota abundant in WWTPs worldwide, using high-quality metagenome-assembled genomes (MAGs) and 16S rRNA amplicon data from 740 Danish and global WWTPs. Many novel taxa were described, encompassing 4 families, 13 genera, and 29 novel species. These were widely distributed across most continents, influenced by factors such as climate zone and WWTP process design. Visualization by fluorescence in situ hybridization (FISH) confirmed their high abundances in many WWTPs based on the amplicon data and showed a filamentous morphology for nearly all species. Most formed thin and short trichomes integrated into the floc structure, unlikely to form the typical inter-floc bridging that hinders activated sludge floc settling. Metabolic reconstruction of 53 high-quality MAGs, representing most of the novel genera, offered further insights into their versatile metabolisms and suggested a primary role in carbon removal and involvement in nitrogen cycling. The presence of glycogen reserves, detected by FISH-Raman microspectroscopy, seemed widespread across the phylum, demonstrating that these bacteria likely utilize glycogen as energy storage to survive periods with limited resources. This study gives a broad overview of the Chloroflexota community in global activated sludge WWTPs and improves our understanding of their roles in these engineered ecosystems. IMPORTANCE Chloroflexota are often abundant members of the biomass in wastewater treatment plants (WWTPs) worldwide, typically with a filamentous morphology, forming the backbones of the activated sludge floc. However, their overgrowth can often cause operational issues connected to poor settling or foaming, impairing effluent quality and increasing operational costs. Despite their importance, few Chloroflexota genera have been characterized so far. Here, we present a comprehensive overview of Chloroflexota abundant in WWTPs worldwide and an in-depth characterization of their morphology, phylogeny, and ecophysiology, obtaining a broad understanding of their ecological role in activated sludge.

Diaphorina citri is the hemipteran pest and vector of a devastating bacterial pathogen of citrus worldwide. In addition to the two core bacterial endosymbionts of D. citri, Candidatus Carsonella ruddii and Candidatus Profftella armatura, the genome of a novel endosymbiont and as of yet undescribed microbe was discovered in a Hawaiian D. citri population through deep sequencing of multiple D. citri populations. Found to be closely related to the genus Asaia in the family Acetobacteraceae by 16S rRNA gene sequence analysis, it forms a sister clade along with other insect-associated 16S rRNA gene sequences from uncultured bacterium found associated with Aedes koreicus and Sogatella furcifera. Multilocus sequence analysis confirmed the phylogenetic placement sister to the Asaia clade. Despite the culturable Asaia clade being the closest phylogenetic neighbour, attempts to culture this newly identified bacterial endosymbiont were unsuccessful. On the basis of these distinct genetic differences, the novel endosymbiont is proposed to be classified into a candidate genus and species ‘Candidatus Kirkpatrickella diaphorinae’. The full genome was deposited in GenBank (accession number CP107052; prokaryotic 16S rRNA OP600170).

Huanglongbing (HLB; also known as citrus greening) is the most destructive bacterial disease of citrus worldwide with no known sustainable cure yet. Herein, we used non-targeted metabolomics and transcriptomics to prove that γ-aminobutyric acid (GABA) accumulation might influence the homeostasis of several metabolic pathways, as well as antioxidant defense machinery, and their metabolism-related genes. Overall, 41 metabolites were detected in ‘Valencia’ sweet orange (Citrus sinensis) leaf extract including 19 proteinogenic amino acids (PAA), 10 organic acids, 5 fatty acids, and 9 other amines (four phenolic amines and three non-PAA). Exogenous GABA application increased most PAA in healthy (except L-threonine, L-glutamine, L-glutamic acid, and L-methionine) and ‘Candidatus L. asiaticus’-infected citrus plants (with no exception). Moreover, GABA accumulation significantly induced L-tryptophan, L-phenylalanine, and α-linolenic acid, the main precursors of auxins, salicylic acid (SA), and jasmonic acid (JA), respectively. Furthermore, GABA supplementation upregulated most, if not all, of amino acids, phenolic amines, phytohormone metabolism-related, and GABA shunt-associated genes in both healthy and ‘Ca. L. asiaticus’-infected leaves. Moreover, although ‘Ca. L. asiaticus’ induced the accumulation of H2O2 and O2•− and generated strong oxidative stress in infected leaves, GABA possibly stimulates the activation of a multilayered antioxidative system to neutralize the deleterious effect of reactive oxygen species (ROS) and maintain redox status within infected leaves. This complex system comprises two major components: (i) the enzymatic antioxidant defense machinery (six POXs, four SODs, and CAT) that serves as the front line in antioxidant defenses, and (ii) the non-enzymatic antioxidant defense machinery (phenolic acids and phenolic amines) that works as a second defense line against ‘Ca. L. asiaticus’-induced ROS in citrus infected leaves. Collectively, our findings suggest that GABA might be a promising alternative eco-friendly strategy that helps citrus trees battle HLB particularly, and other diseases in general.

The devastating citrus disease, Huanglongbing (HLB), is associated with ‘Candidatus Liberibacter sp.’ and transmitted by citrus psyllids. Unfortunately, HLB has no known sustainable cure yet. Herein, we proposed γ-aminobutyric acid (GABA) as a potential eco-friendly therapeutic solution to HLB. Herein, we used GC/MS-based targeted metabolomics combined with gene expression to investigate the role of GABA in citrus response against HLB and to better understand its relationship(s) with different phytohormones. GABA supplementation via root drench boosts the accumulation of endogenous GABA in the leaves of both healthy and ‘Ca. L. asiaticus’-infected trees. GABA accumulation benefits the activation of a multi-layered defensive system via modulating the phytohormone levels and regulating the expression of their biosynthesis genes and some pathogenesis-related proteins (PRs) in both healthy and ‘Ca. L. asiaticus’-infected plants. Moreover, our findings showed that GABA application stimulates auxin biosynthesis in ‘Ca. L. asiaticus’-infected plants via the activation of the indole-3-pyruvate (I3PA) pathway, not via the tryptamine (TAM)-dependent pathway, to enhance the growth of HLB-affected trees. Likewise, GABA accumulation was associated with the upregulation of SA biosynthesis genes, particularly the PAL-dependent route, resulting in higher SA levels that activated CsPR1, CsPR2, CsPR5, and CsWRKY70, which are prominent to activation of the SA-mediated pathway. Additionally, higher GABA levels were correlated with an enhanced JA profile and linked with both CsPR3 and CsPR4, which activates the JA-mediated pathway. Collectively, our findings suggest that exogenous GABA application might be a promising alternative and eco-friendly strategy that helps citrus trees battle HLB.

The anaerobic oxidation of fatty acids and alcohols occurs near the thermodynamic limit of life. This process is driven by syntrophic bacteria that oxidize fatty acids and/or alcohols, their syntrophic partners that consume the products of this oxidation, and the pathways for interspecies electron exchange via these products or direct interspecies electron transfer (DIET). Due to the interdependence of syntrophic microorganisms on each other’s metabolic activity, their isolation in pure cultures is almost impossible. Thus, little is known about their physiology, and the only available way to fill in the knowledge gap on these organisms is genomic and metabolic analysis of syntrophic cultures. Here we report the results of genome sequencing and analysis of an obligately syntrophic alkaliphilic bacterium ‘Candidatus Contubernalis alkaliaceticus’. The genomic data suggest that acetate oxidation is carried out by the Wood–Ljungdahl pathway, while a bimodular respiratory system involving an Rnf complex and a Na+-dependent ATP synthase is used for energy conservation. The predicted genomic ability of ‘Ca. C. alkaliaceticus’ to outperform interspecies electron transfer both indirectly, via H2 or formate, and directly, via pili-like appendages of its syntrophic partner or conductive mineral particles, was experimentally demonstrated. This is the first indication of DIET in the class Dethiobacteria.

AbstractPlant pathogens can alter the behavior of their insect vectors as well as their survival and reproduction. The African psyllid, Trioza erytreae, is one of the vectors of Huanglongbing, a citrus disease caused mainly by “Candidatus Liberibacter asiaticus” (CLas). The purpose of this study was to characterize the effects of CLas on the psyllid, T. erytreae using Citrus volkamerina plants as the study system. The study focused more specifically on the CLas effects prior to and after its acquisition by the psyllid T. erytreae. Our results did not support the hypothesis that CLas effects psyllid probing behavior prior to acquisition; few differences were observed between uninfected T. erytrea feeding on CLas‐infected versus control plants. On the other hand, compared to psyllids that had completed their development on control plants, the ones that had completed their development on a CLas‐infected plant exhibited changes in their behavior (greater velocity), physiology (smaller mass) and biochemistry (lower water and lipid content). Altogether, our results confirm the existence of a marked postacquisition effect on the vector locomotor behavior and a minor preacquisition effect of CLas on the vector behavior, which can be partially explained by physiological and biochemical changes.

Early detection and prompt response are key factors in the eradication of ‘huanglongbing’ (HLB) in California. Currently, qPCR testing of leaf tissue guides the removal of infected trees. However, because of the uneven distribution of ‘Candidatus Liberibacter asiaticus’ (CLas) in an infected tree and asymptomatic infection, selecting the best leaves to sample, from a mature tree with more than 200,000 estimated leaves, is a major hurdle for timely detection. The goal of this study was to address this issue by testing alternative tissues that might improve the CLas detection rate. Using two years of field data, old and young leaves, peduncle bark of fruit, and feeder roots were evaluated for the presence of CLas. Quadrant-peduncle (Q-P) tissue sampling consistently resulted in better CLas detection than any other tissue type. Q-P samples had a 30% higher qPCR positivity rate than quadrant-leaf (Q-L) samples. No significant seasonal patterns were observed. Roots and single peduncles had similar detection rates; both were higher than single leaves or Q-L samples. If symptoms were used to guide sampling, 30% of infected trees would have been missed. Taken together, these results suggest that Q-P tissue sampling is the optimal choice for improved CLas detection under California growing conditions.