Microbiology (medical)

‘Candidatus Liberibacter solanacearum’ (CaLsol) is an uncultured bacterium, transmitted by psyllids and associated with several diseases in Solanaceae and Apiaceae crops. CaLsol detection in psyllids often requires insect destruction, preventing a subsequent morphological identification. In this work, we have assessed the influence on the detection of CaLsol by PCR in Bactericera trigonica (Hemiptera: Psyllidae), of four specimen preparations (entire body, ground, cut-off head, and punctured abdomen) and seven DNA extraction methods (PBS suspension, squashing on membrane, CTAB, Chelex, TRIsureTM, HotSHOT, and DNeasy®). DNA yield and purity ratios, time consumption, cost, and residues generated were also evaluated. Optimum results were obtained through grinding, but it is suggested that destructive procedures are not essential in order to detect CaLsol. Although CaLsol was detected by qPCR with DNA obtained by the different procedures, HotSHOT was the most sensitive method. In terms of time consumption and cost, squashed on membrane, HotSHOT, and PBS were the fastest, while HotSHOT and PBS were the cheapest. In summary, HotSHOT was accurate, fast, simple, and sufficiently sensitive to detect this bacterium within the vector. Additionally, cross-contamination with CaLsol was assessed in the ethanol solutions where B. trigonica specimens were usually collected and preserved. CaLsol-free psyllids were CaLsol-positive after incubation with CaLsol-positive specimens. This work provides a valuable guide when choosing a method to detect CaLsol in vectors according to the purpose of the study.

Anaerobic methanotrophic (ANME) archaea can drive anaerobic oxidation of methane (AOM) using solid iron or manganese oxides as the electron acceptors, hypothetically via direct extracellular electron transfer (EET). This study investigated the response of Candidatus “Methanoperedens nitroreducens TS” (type strain), an ANME archaeon previously characterized to perform nitrate-dependent AOM, to an Fe(III)-amended condition over a prolonged period. Simultaneous consumption of methane and production of dissolved Fe(II) were observed for more than 500 days in the presence of Ca. “M. nitroreducens TS,” indicating that this archaeon can carry out Fe(III)-dependent AOM for a long period. Ca. “M. nitroreducens TS” possesses multiple multiheme c-type cytochromes (MHCs), suggesting that it may have the capability to reduce Fe(III) via EET. Intriguingly, most of these MHCs are orthologous to those identified in Candidatus “Methanoperedens ferrireducens,” an Fe(III)-reducing ANME archaeon. In contrast, the population of Ca. “M. nitroreducens TS” declined and was eventually replaced by Ca. “M. ferrireducens,” implying niche differentiation between these two ANME archaea in the environment.

Anaerobic methanotrophic (ANME) archaea have recently been reported to be capable of using insoluble extracellular electron acceptors via extracellular electron transfer (EET). In this study, we investigated EET by a microbial community dominated by “Candidatus Methanoperedens” archaea at the anode of a bioelectrochemical system (BES) poised at 0 V vs. standard hydrogen electrode (SHE), in this way measuring current as a direct proxy of EET by this community. After inoculation of the BES, the maximum current density was 274 mA m–2 (stable current up to 39 mA m–2). Concomitant conversion of 13CH4 into 13CO2 demonstrated that current production was methane-dependent, with 38% of the current attributed directly to methane supply. Based on the current production and methane uptake in a closed system, the Coulombic efficiency was about 17%. Polarization curves demonstrated that the current was limited by microbial activity at potentials above 0 V. The metatranscriptome of the inoculum was mined for the expression of c-type cytochromes potentially used for EET, which led to the identification of several multiheme c-type cytochrome-encoding genes among the most abundant transcripts in “Ca. Methanoperedens.” Our study provides strong indications of EET in ANME archaea and describes a system in which ANME-mediated EET can be investigated under laboratory conditions, which provides new research opportunities for mechanistic studies and possibly the generation of axenic ANME cultures.

Citrus Huanglongbing (HLB) or citrus greening, is the most destructive disease for citrus worldwide. It is caused by the psyllid-transmitted, phloem-limited bacteria “CandidatusLiberibacter asiaticus” (CLas). To date, there are still no effective practical strategies for curing citrus HLB. Understanding the mechanisms againstCLas can contribute to the development of effective approaches for combatting HLB. However, the unculturable nature ofCLas has hindered elucidating mechanisms againstCLas. In this review, we summarize the main aspects that contribute to the understanding about the mechanisms againstCLas, including (1)CLas virulence targets, focusing on inhibition of virulence genes; (2) activation of citrus host defense genes and metabolites of HLB-tolerant citrus triggered byCLas, and by agents; and (3) we also review the role of citrus microbiome in combattingCLas. Finally, we discuss novel strategies to continue studying mechanisms againstCLas and the relationship of above aspects.

Citrus Huanglongbing (HLB) is the most devastating disease of citrus caused by the Gram-negative phloem-limited bacterium “Candidatus Liberibacter asiaticus” (CLas). It can be transmitted by the Asian citrus psyllid “Diaphorina citri,” by grafting, and by the holoparasitic dodder. In this study, the non-natural host periwinkle (Catharanthus roseus) was infected via dodder (Cuscuta campestris) from CLas-infected citrus plants, and the asymptomatic leaves (AS) were subjected to transcriptomic and small-RNA profiling. The results were analyzed together with a transcriptome dataset from the NCBI repository that included leaves for which symptoms had just occurred (S) and yellowing leaves (Y). There were 3,675 differentially expressed genes (DEGs) identified in AS, and 6,390 more DEGs in S and further 2109 DEGs in Y. These DEGs were commonly enriched in photosystem, chloroplast, membrane, oxidation-reduction process, metal/zinc ion binding on GO. A total of 14,974 DEGs and 336 DE miRNAs (30 conserved and 301 novel) were identified. Through weighted gene co-expression network and nested network analyses, two critical nested miRNA–mRNA regulatory networks were identified with four conserved miRNAs. The primary miR164-NAC1 network is potentially involved in plant defense responses against CLas from the early infection stage to symptom development. The secondary network revealed the regulation of secondary metabolism and nutrient homeostasis through miR828-MYB94/miR1134-HSF4 and miR827-ATG8 regulatory networks, respectively. The findings discovered new potential mechanisms in periwinkle–CLas interactions, and its confirmation can be done in citrus–CLas system later on. The advantages of periwinkle plants in facilitating the quick establishment and greater multiplication of CLas, and shortening latency for disease symptom development make it a great surrogate for further studies, which could expedite our understanding of CLas pathogenesis.

Phytoplasmas are insect-transmitted plant pathogens that cause substantial losses in agriculture. In addition to economic impact, phytoplasmas induce distinct disease symptoms in infected plants, thus attracting attention for research on molecular plant-microbe interactions and plant developmental processes. Due to the difficulty of establishing an axenic culture of these bacteria, culture-independent genome characterization is a crucial tool for phytoplasma research. However, phytoplasma genomes have strong nucleotide composition biases and are repetitive, which make it challenging to produce complete assemblies. In this study, we utilized Illumina and Oxford Nanopore sequencing technologies to obtain the complete genome sequence of ‘Candidatus Phytoplasma luffae’ strain NCHU2019 that is associated with witches’ broom disease of loofah (Luffa aegyptiaca) in Taiwan. The fully assembled circular chromosome is 769 kb in size and is the first representative genome sequence of group 16SrVIII phytoplasmas. Comparative analysis with other phytoplasmas revealed that NCHU2019 has a remarkably repetitive genome, possessing a pair of 75 kb repeats and at least 13 potential mobile units (PMUs) that account for ∼25% of its chromosome. This level of genome repetitiveness is exceptional for bacteria, particularly among obligate pathogens with reduced genomes. Our genus-level analysis of PMUs demonstrated that these phytoplasma-specific mobile genetic elements can be classified into three major types that differ in gene organization and phylogenetic distribution. Notably, PMU abundance explains nearly 80% of the variance in phytoplasma genome sizes, a finding that provides a quantitative estimate for the importance of PMUs in phytoplasma genome variability. Finally, our investigation found that in addition to horizontal gene transfer, PMUs also contribute to intra-genomic duplications of effector genes, which may provide redundancy for subfunctionalization or neofunctionalization. Taken together, this work improves the taxon sampling for phytoplasma genome research and provides novel information regarding the roles of mobile genetic elements in phytoplasma evolution.

Huanglongbing (HLB), caused by “Candidatus liberibacter asiaticus” (CaLas), is one of the most devastating diseases in citrus but its pathogenesis remains poorly understood. Here, we reported the role of the CaLasSDE115 (CLIBASIA_05115) effector, encoded by CaLas, during pathogen-host interactions. Bioinformatics analyses showed that CaLasSDE115 was 100% conserved in all reported CaLas strains but had sequence differences compared with orthologs from other “Candidatus liberibacter.” Prediction of protein structures suggested that the crystal structure of CaLasSDE115 was very close to that of the invasion-related protein B (IalB), a virulence factor from Bartonella henselae. Alkaline phosphatase (PhoA) assay in E. coli further confirmed that CaLasSDE115 was a Sec-dependent secretory protein while subcellular localization analyses in tobacco showed that the mature protein of SDE115 (mSDE115), without its putative Sec-dependent signal peptide, was distributed in the cytoplasm and the nucleus. Expression levels of CaLasSDE115 in CaLas-infected Asian citrus psyllid (ACP) were much higher (∼45-fold) than those in CaLas-infected Wanjincheng oranges, with the expression in symptomatic leaves being significantly higher than that in asymptomatic ones. Additionally, the overexpression of mSDE115 favored CaLas proliferation during the early stages (2 months) of infection while promoting the development of symptoms. Hormone content and gene expression analysis of transgenic plants also suggested that overexpressing mSDE115 modulated the transcriptional regulation of genes involved in systemic acquired resistance (SAR) response. Overall, our data indicated that CaLasSDE115 effector contributed to the early colonization of citrus by the pathogen and worsened the occurrence of Huanglongbing symptoms, thereby providing a theoretical basis for further exploring the pathogenic mechanisms of Huanglongbing disease in citrus.

The considerable economic losses in citrus associated with ‘Candidatus Liberibacter’ and ‘Candidatus Phytoplasma’ presence have alerted all producing regions of the world. In Chile, none of these bacteria have been reported in citrus species. During the years 2017 and 2019, 258 samples presenting symptoms similar to those associated with the presence of these bacteria were examined. No detection of ‘Ca. Liberibacter’ associated with “huanglongbing” disease was obtained in the tested samples; therefore, this quarantine pest is maintained as absent in Chile. However, 14 plants resulted positive for phytoplasmas enclosed in subgroups 16SrV-A (12 plants) and 16SrXIII-F (2 plants). Although they have been found in other plant species, this is the first report of these phytoplasmas in citrus worldwide.