Ecology, Evolution, Behavior and Systematics

Huanglongbing (HLB, or citrus greening disease) affects all citrus varieties worldwide. In the United States, Asia, and South America, the causal agent is ‘ Candidatus Liberibacter asiaticus’ ( CLas), a phloem-limited, uncultured alphaproteobacterium. The hemipteran insect vector Diaphorina citri (Asian citrus psyllid) acquires and transmits CLas in a circulative, propagative manner. In addition to CLas, D. citri hosts multiple symbiotic bacterial species including Wolbachia (wDi). In D. citri, wDi has been sequenced and studied, but specific roles in D. citri biology are unknown. Using well-established quantitative PCR methods, we measured CLas titer in D. citri collected from four groves in central Florida with distinct HLB management strategies and tested whether CLas and wDi titer were correlated in a subset of these insects. Grove site had the largest effect on CLas titer. Sex had no effect on CLas titer, whereas a higher wDi titer was correlated with noninfected insects. Our results suggest that more directed follow-up research is necessary and important to clarify whether field management tactics influence CLas titer in D. citri and to better understand gene-by-environment interactions among D. citri, wDi, and CLas. Now that millions of trees in Florida have been treated with injectable formulations of oxytetracycline, which is likely to decrease bacterial populations in D. citri, this study may represent the last biologically meaningful snapshot of grove-level vector-pathogen ecology in the state during the HLB epidemic.

AbstractThe Candidate Phyla Radiation (CPR) encompasses widespread uncultivated bacteria with reduced genomes and limited metabolic capacities. Most CPR bacteria lack the minimal set of enzymes required for peptidoglycan (PG) synthesis, leaving it unclear how these bacteria produce this essential envelope component. In this study, we analysed the distribution of d‐amino acid racemases that produce the universal PG components d‐glutamate (d‐Glu) or d‐alanine (d‐Ala). We also examined moonlighting enzymes that synthesize d‐Glu or d‐Ala. Unlike other phyla in the domain Bacteria, CPR bacteria do not exhibit these moonlighting activities and have, at most, one gene encoding either a Glu or Ala racemase. One of these ‘orphan’ racemases is a predicted Glu racemase (MurICPR) from the CPR bacterium Candidatus Saccharimonas aalborgenesis. The expression of MurICPR restores the growth of a Salmonella d‐Glu auxotroph lacking its endogenous racemase and results in the substitution of l‐Ala by serine as the first residue in a fraction of the PG stem peptides. In vitro, MurICPR exclusively racemizes Glu as a substrate. Therefore, Ca. Saccharimonas aalborgensis may couple Glu racemization to serine and d‐Glu incorporation into the stem peptide. Our findings provide the first insights into the synthesis of PG by an uncultivated environmental bacterium and illustrate how to experimentally test enzymatic activities from CPR bacteria related to PG metabolism.

ABSTRACT Members of the “ Candidatus Accumulibacter” genus are widely studied as key polyphosphate-accumulating organisms (PAOs) in biological nutrient removal (BNR) facilities performing enhanced biological phosphorus removal (EBPR). This diverse lineage includes 18 “ Ca . Accumulibacter” species, which have been proposed based on the phylogenetic divergence of the polyphosphate kinase 1 ( ppk1 ) gene and genome-scale comparisons of metagenome-assembled genomes (MAGs). Phylogenetic classification based on the 16S rRNA genetic marker has been difficult to attain because most “ Ca . Accumulibacter” MAGs are incomplete and often do not include the rRNA operon. Here, we investigate the “ Ca . Accumulibacter” diversity in pilot-scale treatment trains performing BNR under low dissolved oxygen (DO) conditions using genome-resolved metagenomics. Using long-read sequencing, we recovered medium- and high-quality MAGs for 5 of the 18 “ Ca . Accumulibacter” species, all with rRNA operons assembled, which allowed a reassessment of the 16S rRNA-based phylogeny of this genus and an analysis of phylogeny based on the 23S rRNA gene. In addition, we recovered a cluster of MAGs that based on 16S rRNA, 23S rRNA, ppk1 , and genome-scale phylogenetic analyses do not belong to any of the currently recognized “ Ca . Accumulibacter” species for which we propose the new species designation “ Ca . Accumulibacter jenkinsii” sp. nov. Relative abundance evaluations of the genus across all pilot plant operations revealed that regardless of the operational mode, “ Ca . A. necessarius” and “ Ca . A. propinquus” accounted for more than 40% of the “ Ca . Accumulibacter” community, whereas the newly proposed “ Ca . A. jenkinsii” accounted for about 5% of the “ Ca . Accumulibacter” community. IMPORTANCE One of the main drivers of energy use and operational costs in activated sludge processes is the amount of oxygen provided to enable biological phosphorus and nitrogen removal. Wastewater treatment facilities are increasingly considering reduced aeration to decrease energy consumption, and whereas successful BNR has been demonstrated in systems with minimal aeration, an adequate understanding of the microbial communities that facilitate nutrient removal under these conditions is still lacking. In this study, we used genome-resolved metagenomics to evaluate the diversity of the “ Candidatus Accumulibacter” genus in pilot-scale plants operating with minimal aeration. We identified the “ Ca . Accumulibacter” species enriched under these conditions, including one novel species for which we propose “ Ca . Accumulibacter jenkinsii” sp. nov. as its designation. Furthermore, the MAGs obtained for five additional “ Ca . Accumulibacter” species further refine the phylogeny of the “ Ca . Accumulibacter” genus and provide new insight into its diversity within unconventional biological nutrient removal systems.

AbstractThe unicellular cyanobacterium Candidatus Atelocyanobacterium thalassa (UCYN‐A) is a key diazotroph in the global ocean owing to its high N2 fixation rates and wide distribution in marine environments. Nevertheless, little is known about UCYN‐A in oxygen‐deficient zones (ODZs), which may be optimal environments for marine diazotrophy. Therefore, the distribution and diversity of UCYN‐A were studied in two consecutive years under contrasting phases (La Niña vs. El Niño) of El Niño Southern Oscillation (ENSO) along a transect in the ODZ of the Mexican Pacific upwelling system. Of the three UCYN‐A sublineages found, UCYN‐A1 and UCYN‐A3 were barely detected, whereas UCYN‐A2 was dominant in all the stations and showed a wide distribution in both ENSO phases. The presence of UCYN‐A was associated with well‐oxygenated waters, but it was also found for the first time under suboxic conditions (<20 μM) at the bottom of a shallow coastal station, within the oxygen‐poor and nutrient‐rich Subsurface Subtropical water mass. This study contributes to the understanding of UCYN‐A distribution under different oceanographic conditions associated with ENSO phases in upwelling systems, especially because of the current climate change and increasing deoxygenation in many areas of the world's oceans.

<p><strong>Background.</strong> Climate change models have projected an increase in the distribution of certain crop pests of economic importance by forecasting more favorable future conditions for these organisms. In citrus farming, Huanglongbing is one of the most devastating diseases worldwide, since it has caused the death of millions of trees. <strong>Objetive.</strong> The objective of this study was to estimate the current and future distribution of <em>Candidatus </em>Liberibacter asiaticus in Mexico, under the climate change scenarios SSP2 4.5 and SSP5 8.5, for the years 2050 and 2070. <strong>Methodology.</strong> Distribution models were generated with MaxEnt and R, using uncorrelated bioclimatic variables from eight General Circulation Models (GCM) derived from CMIP6 and disease presence data. <strong>Results.</strong> The results indicate that the current suitability is 44.6 %. The future distribution depended on how model predictions were pooled. An optimistic approach that considered the intersection of all models showed a small reduction of 4.1% while, considering the union of all the GCM models, the increase will vary from 12.3 to 20.1 % of the Mexican territory depending on the particular scenario and time projection. <strong>Implications.</strong> The zones of potential occurrence of <em>Candidatus </em>Liberibacter asiaticus include most of the citrus-growing areas in Mexico. <strong>Conclusion. </strong>In some regions, future scenarios show a reduction in the potential occurrence of the species in citrus plantations. However, the risk remains because its surroundings include suitable areas that can be sources of dissemination of the disease. </p>

‘Candidatus Phytoplasma ulmi’ (Ca. P. ulmi) belongs to the ribosomal subgroup 16SrV-A and is associated with dieback, shoot proliferation and yellows disease on various Ulmus spp. Other plant species, such as Carpinus betulus and Prunus spp. have also been reported infected by the same pathogen. In 2021, in the frame of research activities focused on grapevine’s Flavescence dorée (FD), one specimen of Orientus ishidae - an East Palearctic leafhopper that was identified as an alternative vector of FD phytoplasmas - was found harboring Ca. P. ulmi in southern Switzerland. No phytoplasmas were detected in plant samples taken in the same location. Orientus ishidae has already been reported to be able to acquire diverse phytoplasmas associated with other plant diseases, such as Peach X-disease. This is the first report of Ca. P. ulmi in Switzerland, as well as in O. ishidae. Ca. P. ulmi may potentially be present in the wild compartment of the Swiss Pre-alpine and Alpine range, but no dedicated survey has so far been conducted. In the case of O. ishidae, this finding highlights the broad affinity of such a species for the acquisition of several phytoplasmas. This calls for a further investigation regarding its potential role as a vector on various pathosystems of agronomic importance.

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. The phloem-restricted bacterium Candidatus Liberibacter asiaticus (CLas) is considered to be the main pathogen responsible for HLB. There is currently no effective practical strategy for the control of HLB. Our understanding of how pathogens cause HLB is limited because CLas has not been artificially cultured. In this study, 15 potential virulence factors were predicted from the proteome of CLas through DeepVF and PHI-base searches. One among them, FlgI, was found to inhibit yeast growth when expressed in Saccharomyces cerevisiae. The expression of the signal peptide of FlgI fused with PhoA in Escherichia coli resulted in the discovery that FlgI was a novel Sec-dependent secretory protein. We further found that the carboxyl-terminal HA-tagged FlgI was secreted via outer membrane vesicles in Sinorhizobium meliloti. Fluoresence localization of transient expression FlgI-GFP in Nicotiana benthamiana revealed that FlgI is mainly localized in the cytoplasm, cell periphery, and nuclear periphery of tobacco cells. In addition, our experimental results suggest that FlgI has a strong ability to induce callose deposition and cell necrosis in N. benthamiana. Finally, by screening a large library of compounds in a high-throughput format, we found that cyclosporin A restored the growth of FlgI-expressing yeast. These results confirm that FlgI is a novel Sec-dependent effector, enriching our understanding of CLas pathogenicity and helping to develop new and more effective strategies to manage HLB.