Publications

4497

‘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.

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 .

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.

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.

Huanglongbing, a globally devastating citrus disease, is associated with Candidatus Liberibacter asiaticus (CLas) and is mainly transmitted by Diaphorina citri. Verification of the distribution and dynamics of CLas in D. citri is critical to understanding CLas transmitted by vectors in nature. Here, the distribution and titers of CLas in different sexes and tissues of D. citri adults were investigated by fluorescence in-situ hybridization (FISH) and quantitative real-time PCR (qRT-PCR). Results showed that CLas had widespread distribution in the brain, salivary glands, digestive system, and reproductive system of both females and males, indicating a systemic infection of CLas in D. citri. Moreover, CLas fluorescence intensity and titers were significantly increased in both the digestive system and the female reproductive system with development and there was a marked decreased in both the salivary glands and the male brain, but there was no significant change in the female brain or the male reproductive system. Furthermore, the distribution and dynamics of CLas in embryos and nymphs were investigated. CLas was observed in all laid eggs and subsequent first–second-instar nymphs, indicating that a high percentage of embryos and nymphs resulting from infected D. citri mothers were infected with CLas.

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.

We report sequences of the complete linear chromosome and five linear plasmids of the relapsing fever spirochete “ Candidatus Borrelia fainii” Qtaro. The chromosome sequence of 951,861 bp and the 243,291 bp of plasmid sequences were predicted to contain 852 and 239 protein-coding genes, respectively. The predicted total GC content was 28.4%.

The phytoplasma “ Candidatus Phytoplasma rubi” is associated with Rubus stunt disease. The complete genome was determined by assembling Oxford Nanopore Technologies system-derived long reads, with short-read polishing with Illumina reads. The genome of strain RS, from Germany, is organized in one circular chromosome with a length of 762 kb.

AbstractPhytoplasmas are obligate cell wall-less prokaryotic bacteria that primarily multiply in plant phloem tissue. Jujube witches’ broom (JWB) associated with phytoplasma is a destructive disease of jujube (Ziziphus jujuba Mill.). Here we report the complete ‘Candidatus Phytoplasma ziziphi’ chromosome of strain Hebei-2018, which is a circular genome of 764,108-base pairs with 735 predicted CDS. Notably, extra 19,825 bp (from 621,995 to 641,819 bp) compared to the previously reported one complements the genes involved in glycolysis, such as pdhA, pdhB, pdhC, pdhD, ackA, pduL and LDH. The synonymous codon usage bias (CUB) patterns by using comparative genomics analysis among the 9 phytoplasmas were similar for most codons. The ENc-GC3s analysis among the 9 phytoplasmas showed a greater effect under the selection on the CUBs of phytoplasmas genes than mutation and other factors. The genome exhibited a strongly reduced ability in metabolic synthesis, while the genes encoding transporter systems were well developed. The genes involved in sec-dependent protein translocation system were also identified.The expressions of nine FtsHs encoding membrane associated ATP-dependent Zn proteases and Mn-SodA with redox capacity in the Ca. P. ziziphi was positively correlated with the phytoplasma concentration. Taken together, the genome will not only expand the number of phytoplasma species and provide some new information about Ca. P. ziziphi, but also contribute to exploring its pathogenic mechanism.