Publications

4502

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.”

‘ Candidatus Liberibacter asiaticus’ (CLas) is associated with the devastating citrus disease Huanglongbing (HLB). Young flushes are the center of the HLB pathosystem due to their roles in the psyllid life cycle and in the acquisition and transmission of CLas. However, the early events of CLas infection and how CLas modulates young flush physiology remain poorly understood. Here, transmission electron microscopy analysis showed that the mean diameter of the sieve pores decreased in young leaves of HLB-positive trees after CLas infection, consistent with CLas-triggered callose deposition. RNA-seq-based global expression analysis of young leaves of HLB-positive sweet orange with (CLas-Pos) and without (CLas-Neg) detectable CLas demonstrated a significant impact on gene expression in young leaves, including on the expression of genes involved in host immunity, stress response, and plant hormone biosynthesis and signaling. CLas-Pos and CLas-Neg expression data displayed distinct patterns. The number of upregulated genes was higher than that of the downregulated genes in CLas-Pos for plant−pathogen interactions, glutathione metabolism, peroxisome, and calcium signaling, which are commonly associated with pathogen infections, compared with the healthy control. On the contrary, the number of upregulated genes was lower than that of the downregulated genes in CLas-Neg for genes involved in plant−pathogen interactions and peroxisome biogenesis/metabolism. Additionally, a time-course quantitative reverse transcription-PCR-based expression analysis visualized the induced expression of companion cell-specific genes, phloem protein 2 genes, and sucrose transport genes in young flushes triggered by CLas. This study advances our understanding of early events during CLas infection of citrus young flushes.

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.