Publications

4478

Huanglongbing (HLB), the most destructive citrus disease, is associated with unculturable, phloem-limited Candidatus Liberibacter species, mainly Ca. L. asiaticus (Las). Las is transmitted naturally by the insect Diaphorina citri. In a previous study, we determined that the Oceanian citrus relatives Eremocitrus glauca, Microcitrus warburgiana, Microcitrus papuana, and Microcitrus australis and three hybrids among them and Citrus were full-resistant to Las. After 2 years of evaluations, leaves of those seven genotypes remained Las-free even with their susceptible rootstock being infected. However, Las was detected in their stem bark above the scion-rootstock graft union. Aiming to gain an understanding of the full-resistance phenotype, new experiments were carried out with the challenge-inoculated Oceanian citrus genotypes through which we evaluated: (1) Las acquisition by D. citri fed onto them; (2) Las infection in sweet orange plants grafted with bark or budwood from them; (3) Las infection in sweet orange plants top-grafted onto them; (4) Las infection in new shoots from rooted plants of them; and (5) Las infection in new shoots of them after drastic back-pruning. Overall, results showed that insects that fed on plants from the Oceanian citrus genotypes, their canopies, new flushes, and leaves from rooted cuttings evaluated remained quantitative real-time polymerase chain reaction (qPCR)-negative. Moreover, their budwood pieces were unable to infect sweet orange through grafting. Furthermore, sweet orange control leaves resulted infected when insects fed onto them and graft-receptor susceptible plants. Genomic and morphological analysis of the Oceanian genotypes corroborated that E. glauca and M. warburgiana are pure species while our M. australis accession is an M. australis × M. inodora hybrid and M. papuana is probably a M. papuana × M. warburgiana hybrid. E. glauca × C. sinensis hybrid was found coming from a cross between E. glauca and mandarin or tangor. Eremocitrus × Microcitrus hybrid is a complex admixture of M. australasica, M. australis, and E. glauca while the last hybrid is an M. australasica × M. australis admixture. Confirmation of consistent full resistance in these genotypes with proper validation of their genomic parentages is essential to map properly genomic regions for breeding programs aimed to generate new Citrus-like cultivars yielding immunity to HLB.

Abstract The candidate phyla radiation (CPR) has been described as an obligatory group of ultrasmall bacteria associated with host bacteria. They phylogenetically represent a subdivision of bacteria distinct from other living organisms. Using polyphasic approaches, we screened human faecal samples for the detection of Saccharibacteria. The new sequences obtained by sequencing were compared to the complete CPR genomes available to date. Then, we attempted a co-culture of CPR-bacteria and non-CPR bacteria from human faecal samples. We finally aimed to evaluate the prevalence and distribution of these Saccharibacteria sequences in human sources in 16S amplicon datasets. We were able to reconstitute two high-quality Saccharibacteria genomes named Minimicrobia massiliensis and Minimicrobia timonensis. We have established, for the first time in human digestive samples, the coculture of Candidatus Saccharibacteria with two different bacterial hosts. Finally, we showed that 12.8% (610/4,756) of samples sequenced in our laboratory were positive for operational taxonomic units (OTUs) assigned to M.massiliensis. and significantly enriched in human respiratory and oral microbiota. Here, we reported the first genomes and coculture of Saccharibacteria from human gut specimens. This study opens a new field, particularly in the study of the involvement of CPR in the human intestinal microbiota.

“Candidatus Liberibacter asiaticus” (CLas) is the causal agent of citrus Huanglongbing (HLB, also called citrus greening disease), a highly destructive disease threatening citrus production worldwide. A novel Microviridae phage (named CLasMV1) has been found to infect CLas, providing a potential therapeutic strategy for CLas/HLB control. However, little is known about the CLasMV1 biology. In this study, we analyzed the population dynamics of CLasMV1 between the insect vector of CLas, the Asian citrus psyllid (ACP, Diaphorina citri Kuwayama) and the holoparasitic dodder plant (Cuscuta campestris Yunck.); both acquired CLasMV1-infected CLas from an HLB citrus. All CLas-positive dodder samples were CLasMV1-positive, whereas only 32% of CLas-positive ACP samples were identified as CLasMV1-positive. Quantitative analyses showed a similar distribution pattern of CLasMV1 phage and CLas among eight citrus cultivars by presenting at highest abundance in the fruit pith and/or the center axis of the fruit. Transcriptome analyses revealed the possible lytic activity of CLasMV1 on CLas in fruit pith as evidenced by high-level expressions of CLasMV1 genes, and CLas genes related to cell wall biogenesis and remodeling to maintain the CLas cell envelope integrity. The up-regulation of CLas genes were involved in restriction–modification system that could involve possible phage resistance for CLas during CLasMV1 infection. In addition, the regulation of CLas genes involved in cell surface components and Sec pathway by CLasMV1 phage could be beneficial for phage infection. This study expanded our knowledge of CLasMV1 phage that will benefit further CLas phage research and HLB control.

AbstractZebra chip disease (ZC), associated with the plant pathogenic bacterium ‘Candidatus Liberibacter solanacearum’ (psyllaurous) (CLso), is a major threat to global potato production. In addition to yield loss, CLso infection causes discoloration in the tubers, rendering them unmarketable. CLso is transmitted by the potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae). ZC is managed by prophylactic insecticide applications to control the vector, which is costly and carries environmental and human health risks. Given the expense, difficulty, and unsustainability of managing vector‐borne diseases with insecticides, identifying sources of resistance to CLso and developing varieties that are resistant or tolerant to CLso and/or potato psyllids has become a major goal of breeding efforts. These efforts include field and laboratory evaluations of noncultivated germplasm and cultivars, studies of tubers in cold storage, detailed quantifications of biochemical responses to infection with CLso, possible mechanisms underlying insect resistance, and traditional examination of potato quality following infections. This review provides a brief history of ZC and potato psyllid, a summary of currently available tools to manage ZC, and a comprehensive review of breeding efforts for ZC and potato psyllid management within the greater context of Integrated Pest Management (IPM) strategies. © 2022 Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Summary Cultivation via classical agar plate (CAP) approaches is widely used to study microbial communities, but they are time‐consuming. An alternative approach is the application of single‐cell dispensing (SCD), which allows high‐throughput, label‐free sorting of microscopic particles. We aimed to develop a new anaerobic SCD workflow to cultivate human gut bacteria and compared it with CAP using faecal communities on three rich culture media. We found that the SCD approach significantly decreased the experimental time to obtain pure cultures from 17 ± 4 to 5 ± 0 days, while the isolate diversity and relative abundance coverage were comparable for both approaches. We further tested the total captured fraction by sequencing the sorted bacteria directly after growth as bulk biomass from 2400 dispensed single cells without downstream identification of individual strains. In this approach, the cultured fraction increased from 35.2% to 52.2% for SCD, highlighting the potential for deeper cultivation projects from single samples. SCD‐based cultivation also captured species not detected by sequencing (16 ± 5 per sample, including seven novel taxa). From this work, 82 human gut bacterial species across five phyla (Actinobacteriota, Bacteroidota, Desulfobacterota, Firmicutes and Proteobacteria) and 24 families were obtained, including the first cultured member of 11 novel genera and 10 novel species that were fully characterized taxonomically.