Publications

4795

‘Candidatus Liberibacter asiaticus’ is the bacterium associated with the citrus disease known as huanglongbing (HLB). This study evaluated the influence of ‘Ca. L. asiaticus’ infection on a number of key plant physiological variables concerning photosynthesis, cell integrity, reactive oxygen species scavengers’ activity, and osmoregulation of two different species of citrus—the pomelo Citrus maxima and the mandarin C. reticulata ‘Tankan’—relative to their measured ‘Ca. L. asiaticus’ infection load. Results indicated that all measured physiological variables except soluble sugar were affected by increased ‘Ca. L. asiaticus’ infection titers, wherein the variety C. maxima proved overall more resistant than C. reticulata. ‘Ca. L. asiaticus’ infection was linked in both plants to decrease in chlorophyll concentration, cell membrane permeability, and malondialdehyde, as well as increased free proline and starch contents. Chlorophyll fluorescence measurements taken 9 months after grafting the mandarin C. reticulata with ‘Ca. L. asiaticus’ scions revealed a significant decrease in the photosynthesis variables maximum photochemical quantum yield of photosystem II (PSII), effective photochemical quantum yield of PSII, and coefficient of photochemical fluorescence quenching assuming interconnected PSII antennae, whereas nonphotochemical fluorescence quenching increased significantly; C. maxima plants, on the other hand, did not show significant differences until the 12th month from infection exposure. The variables superoxide dismutase, catalase, peroxidase, and soluble protein initially increased and later decreased. In addition, progression of ‘Ca. L. asiaticus’ replication in both citrus species was accompanied by rapid changes in three reactive oxygen species scavenging enzymes in C. maxima, while the pattern was different in C. reticulata. We hypothesize that the observed interspecific differences in physiological change are related to their relative resistance against ‘Ca. L. asiaticus’ infection. These results provide a scaffold for better describing the pathogenesis, selecting the most resistant breeds, or even validating pertaining omics research; ultimately, these detailed observations can facilitate the diagnosis of ‘Ca. L. asiaticus’ infection.

Abstract CVPD disease (Huanglongbing) is the most severe citrus disease caused by the bacterium Cand. Liberibacter asiaticus. This pathogen lives in the phloem tissue of citrus plants and is transmitted through the vector Diaphorina citri and by grafting. The study aimed to know the presence of CVPD disease in Orange cv Selayar nurseries and mother tree gardens in the Selayar Islands Regency. The PCR test was carried out in the research using the Laboratory of Agricultural Biotechnology, Hasanuddin University. The DNA of the citrus leaves was extracted using the Genomic DNA Kit method and the DNA CLas was amplified using the polymerase chain reaction (PCR) with the primer pairs OI1-O12c. The results showed that the percentage of mother plants of orange cv. Selayar infected with CVPD-like symptoms were 10-21 %. The percentage of citrus seedlings with CVPD-like symptoms in 2 nurseries were 0.27% and 0.09%. The symptoms of CVPD disease have been confirmed using a PCR technique, which was successful in amplifying the DNA fragment of CLa measuring 1160 bp, although not all symptoms suspected of CVPD are confirmed positive.

AbstractMethanogenic and methanotrophic archaea produce and consume the greenhouse gas methane, respectively, using the reversible enzyme methyl-coenzyme M reductase (Mcr). Recently, Mcr variants that can activate multicarbon alkanes have been recovered from archaeal enrichment cultures. These enzymes, called alkyl-coenzyme M reductase (Acrs), are widespread in the environment but remain poorly understood. Here we produced anoxic cultures degrading mid-chain petroleum n-alkanes between pentane (C5) and tetradecane (C14) at 70 °C using oil-rich Guaymas Basin sediments. In these cultures, archaea of the genus Candidatus Alkanophaga activate the alkanes with Acrs and completely oxidize the alkyl groups to CO2. Ca. Alkanophaga form a deep-branching sister clade to the methanotrophs ANME-1 and are closely related to the short-chain alkane oxidizers Ca. Syntrophoarchaeum. Incapable of sulfate reduction, Ca. Alkanophaga shuttle electrons released from alkane oxidation to the sulfate-reducing Ca. Thermodesulfobacterium syntrophicum. These syntrophic consortia are potential key players in petroleum degradation in heated oil reservoirs.

An updated real-time multiplex quantitative polymerase chain reaction (qPCR) assay was designed and validated for the simultaneous detection of three ‘ Candidatus Liberibacter species’ (CLsp), ‘ Ca. Liberibacter asiaticus’ (CLas), ‘africanus’ (CLaf), and ‘americanus’ (CLam), associated with the huanglongbing disease of citrus. The multiplex assay was designed based on the qPCR assay published in 2006 by Li et al., considering all available CLsp 16S rRNA gene sequences in GenBank and the MIQE guidelines and workflow for qPCR optimization, which became available after 2006. When using the updated multiplex CLsp qPCR assay compared with singleplex qPCR, no significant increase in quantitative cycle (Cq) values was detected. The specificity and sensitivity of the updated qPCR assay was optimal, and measuring the intra- and interassay variations confirmed the reproducibility and repeatability of the assay. The assay was also successfully used with a large number of diverse samples at independent laboratories in four countries, thus demonstrating its transferability, applicability, practicability, and robustness as different qPCR reaction conditions or instruments had a minor effect on Cq values. This updated multiplex CLsp qPCR assay can be used in a variety of citrus surveys, germplasm, or nursery stock programs that require different pathogen detection tools for their successful operation. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

AbstractMany Rickettsia species of the spotted fever group (SFG) cause tick‐borne diseases known as “spotted fever.” One of the candidate SFG Rickettsia species is “Candidatus Rickettsia kotlanii,” which was first detected in Haemaphysalis concinna in Hungary in 2006. However, its precise phylogenetic position in the SFG is not clear because only single‐gene sequence–based phylogenetic analyses were performed using very limited genes. Here, we present the complete genome sequences of two Japanese “Ca. R. kotlanii” isolates, which differed only by a 135 bp insertion/deletion (InDel). Using these genomes and publicly available whole genome sequences of other Rickettsia species, the precise phylogenetic position of “Ca. R. kotlanii” in Rickettsia was determined to be in a clade of the SFG. The phylogenetic relationships and average nucleotide identity of “Ca. R. kotlanii” relative to the other species indicated that “Ca. R. kotlanii” is an independent taxon in the SFG. Notably, although the genomes of the two isolates were almost identical, the isolates were obtained from different tick species in different regions and years, suggesting extremely low genomic diversity in “Ca. R. kotlanii.” While the genome of “Ca. R. kotlanii” is the smallest in the transitional group and SFG Rickettsia sequenced to date, we identified genes uniquely present or absent in “Ca. R. kotlanii,” but most were apparently degraded. Therefore, analyses of differences at the sequence (single nucleotide polymorphisms and small InDels) or gene expression level will be required to understand the functional or physiological features unique to “Ca. R. kotlanii.”

An anaerobic microbial enrichment culture was used to study members of candidate phyla that are difficult to grow in the lab. We were able to visualize tiny “ Candidatus Nealsonbacteria” cells attached to a large Methanothrix cell, revealing a novel episymbiosis.

ABSTRACTEnterococci are commensal gut microbes of most land animals. They diversified over hundreds of millions of years adapting to evolving hosts and host diets. Of over 60 known enterococcal species,Enterococcus faecalisandE. faeciumuniquely emerged in the antibiotic era among leading causes of multidrug resistant hospital-associated infection. The basis for the association of particular enterococcal species with a host is largely unknown. To begin deciphering enterococcal species traits that drive host association, and to assess the pool ofEnterococcus-adapted genes from which known facile gene exchangers such asE. faecalisandE. faeciummay draw, we collected 886 enterococcal strains from nearly 1,000 specimens representing widely diverse hosts, ecologies and geographies. This provided data on the global occurrence and host associations of known species, identifying 18 new species in the process expanding genus diversity by >25%. The novel species harbor diverse genes associated with toxins, detoxification, and resource acquisition.E. faecalisandE. faeciumwere isolated from a wide diversity of hosts highlighting their generalist properties, whereas most other species exhibited more restricted distributions indicative of specialized host associations. The expanded species diversity permitted theEnterococcusgenus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades as well as genes associated with range expansion, such as B-vitamin biosynthesis and flagellar motility. Collectively, this work provides an unprecedentedly broad and deep view of the genusEnterococcus, potential threats to human health, and new insights into its evolution.SIGNIFICANCEEnterococci, host-associated microbes that are now leading drug-resistant hospital pathogens, arose as animals colonized land over 400 million years ago. To globally assess the diversity of enterococci now associated with land animals, we collected 886 enterococcal specimens from a wide range of geographies and ecologies, ranging from urban environments to remote areas generally inaccessible to humans. Species determination and genome analysis revealed host associations from generalists to specialists, and identified 18 new species, increasing the genus by over 25%. This added diversity provided greater resolution of the genus clade structure, identifying new features associated with species radiations. Moreover, the high rate of new species discovery shows that tremendous genetic diversity in Enterococcus remains to be discovered.