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Members of the class Opitutae are widely distributed in various environments such as rice paddy soil, freshwater lakes, seawater, marine sediment, and invertebrate digestive tracts. The class currently consists of two orders, Opitutales and Puniceicoccales, represented by the families Opitutaceae and Puniceicoccaceae, respectively, which are primarily delineated on the basis of 16S rRNA gene sequences and limited phenotypic characterizations of a few type strains. The scarcity of 16S rRNA gene and genome sequences generated from the type strains of the class Opitutae constrained our understanding of the ecological distribution and adequate resolution of its taxonomy. Here, an Opitutae strain designated WMMB3T, isolated from a mangrove sediment, was subjected to taxonomic characterization. The 16S rRNA gene of strain WMMB3T shared high sequence similarities with Coraliomargarita akajimensis DSM 45221T and C. sinensis WN38T of 96.1 and 95.9%, respectively. Phylogenetic analysis suggested that strain WMMB3T formed a monophyletic branch affiliated to the genus Coraliomargarita. The average nucleotide identity (ANI) values, digital DNA–DNA hybridization (dDDH) values and average amino acid identity (AAI) values of strain WMMB3T compared between Coraliomargarita members were 71.8–72.5, 20.7, and 68.2–68.7%, respectively, indicating that strain WMMB3T represented a novel species of Coraliomargarita. The genome of strain WMMB3T was 4.5 Mbp with a DNA G + C content of 56.0%. The respiratory quinone was menaquinone-7. The major fatty acids were iso-C14:0, and C18:1ω9c. Based on genomic, phenotypic, and chemotaxonomic characterizations, strain WMMB3T represents a novel species, and Coraliomargarita parva sp. nov. is proposed. Additionally, the phylogenomic analysis of more than 500 genomes of the class Opitutae, encompassing a majority of uncultivated bacteria and a few type strains, was performed using the Genome Taxonomic Database toolkit (GTDB-Tk) to present adequate resolution of the taxonomy. Combined with 16S rRNA gene sequence phylogeny and genomic relatedness, five novel families retrieved mainly from marine habitats were proposed: Coraliomargaritaceae fam. nov., Pelagicoccaceae fam. nov., Cerasicoccaeae fam. nov., Oceanipulchritudinaceae fam. nov., and Alterococcaeae fam. nov. AAI values of 58–60% could be considered as the boundary to delineate families of the class Opitutae. This study provided a new taxonomic framework of the class Opitutae based on the genomic data.

Huanglongbing (HLB), also known as citrus greening, is an insidious disease in citrus and has become a threat to the sustainability of the citrus industry worldwide. In the U.S., Candidatus Liberibacter asiaticus (CLas) is the pathogen that is associated with HLB, an unculturable, phloem-limited bacteria, vectored by the Asian Citrus Psyllid (ACP, Diaphorina citri). There is no known cure nor treatment to effectively control HLB, and current control methods are primarily based on the use of insecticides and antibiotics, where effectiveness is limited and may have negative impacts on beneficial and non-target organisms. Thus, there is an urgent need for the development of effective and sustainable treatment options to reduce or eliminate CLas from infected trees. In the present study, we screened citrus-derived endophytes, their cell-free culture supernatants (CFCS), and crude plant extracts for antimicrobial activity against two culturable surrogates of CLas, Sinorhizobium meliloti and Liberibacter crescens. Candidates considered high-potential antimicrobial agents were assessed directly against CLas in vitro, using a propidium monoazide–based assay. As compared to the negative controls, statistically significant reductions of viable CLas cells were observed for each of the five bacterial CFCS. Subsequent 16S rRNA gene sequencing revealed that each of the five bacterial isolates were most closely related to Bacillus amyloliquefaciens, a species dominating the market of biological control products. As such, the aboveground endosphere of asymptomatic survivor citrus trees, grown in an organic orchard, were found to host bacterial endophytes capable of effectively disrupting CLas cell membranes. These results concur with the theory that native members of the citrus microbiome play a role in the development of HLB. Here, we identify five strains of Bacillus amyloliquefaciens demonstrating notable potential to be used as sources of novel antimicrobials for the sustainable management of HLB.

Huanglongbing (HLB) is a devastating disease that affects all commercial citrus species worldwide. The disease is associated with bacteria of three species of the genus ‘Candidatus Liberibacter’ transmitted by psyllid vectors. To date, HLB has no cure, so preventing its introduction into HLB-free areas is the best strategy to control its spread. For that, the use of accurate, sensitive, specific, and reliable detection methods is critical for good integrated management of this serious disease. This study presents a new real-time recombinase polymerase amplification (RPA) protocol able to detect the three ‘Ca. Liberibacter’ species associated with HLB in both plant and insect samples, validated according to European and Mediterranean Plant Protection Organization (EPPO) guidelines and tested on 365 samples from nine different geographic origins. This new protocol does not require nucleic acid purification or specialized equipment, making it ideal to be used under field conditions. It is based on specific primers and probe targeting a region of fusA gene, which shows a specificity of 94%–100%, both in silico and in vitro, for the ‘Ca. Liberibacter’ species associated with HLB. The analytical sensitivity of the new protocol is excellent, with a reliable detection limit in the order of 101 copies per microliter in HLB-infected plant and insect material. The repeatability and reproducibility of the new methods showed consistent results. Diagnostic parameters of the new RPA protocol were calculated and compared with the gold standard technique, a quantitative real-time PCR, in both crude extracts of citrus plants and insect vectors. The agreement between the two techniques was almost perfect according to the estimated Cohen’s kappa index, with a diagnostic sensitivity and specificity of 83.89% and 100%, respectively, and a relative accuracy of 91.59%. Moreover, the results are obtained in less than 35 min. All these results indicate the potential of this new RPA protocol to be implemented as a reliable on-site detection kit for HLB due to its simplicity, speed, and portability.

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.

AbstractA 9‐year‐old neutered male cat, previously test‐positive for feline immunodeficiency virus (FIV), was presented with an history of vomiting, hyporexia, and weight loss. Panleukopenia was identified on complete blood counts, and bone marrow evaluation revealed ineffective granulocytic hyperplasia and rare neutro‐, erythro‐, and rubriphagocytosis. Prednisolone was initiated with no response, and progression to pancytopenia occurred. On abdominal ultrasonographic examination, splenomegaly was present. PCR testing was positive for Candidatus Mycoplasma haemominutum and IgG antibodies against Toxoplasma gondii were detected (titer 1:2560). Treatment with antibiotics, feline recombinant interferon‐ω, chlorambucil, mycophenolate, and raltegravir was implemented with no clinical improvement, and splenectomy was performed. Cytologic evaluation of splenic aspirates revealed exuberant neutro‐, erythro‐, and rubriphagocytosis. Histopathology of the spleen also showed many erythrophagocytic macrophages with no evidence of malignancy, and a diagnosis of hemophagocytic syndrome (HS) was made. The WBC count and hematocrit reached reference values 1 day and 3 months, respectively, after splenectomy. The cat was treated with cyclosporine and lomustine. Disease progression led to the development of septic hepatitis, and the cat was euthanized. To our knowledge, this is the first case of presumptive HS in cats that might have been associated with FIV, Toxoplasma gondii, and Candidatus Mycoplasma haemominutum co‐infection.

‘Candidatus Liberibacter solanacearum’ (Lso) is the causal agent of zebra chip of potato (Solanum tuberosum), which can significantly reduce potato yield. In this study, a loop-mediated isothermal amplification (LAMP) method for the detection of Lso haplotypes A and B was developed and evaluated. Two sets of LAMP primers named LAMP-A and LAMP-B were designed and tested for specificity and sensitivity. Both LAMP-A and LAMP-B were specific to Lso in in silico analysis using the Primer-Blast tool. The LAMP-A and LAMP-B could only produce positive signals from DNA mixtures of Lso-infected tomato but not from the genomic DNA of 37 nontarget plant pathogens. The sensitivity of LAMP-A and LAMP-B on Lso haplotypes A and B were tested on gBlocks and genomic DNA from Lso-infected tomato. On the genomic DNA for LAMP-A, the lowest amount of template DNA for a positive LAMP reaction was 2 to 20 ng on four haplotype A strains and 20 to 80 ng on four haplotype B strains; for LAMP-B, the lowest amount of template DNA for a positive LAMP reaction was 0.02 to 2 ng on four haplotype B strains and 20 ng to no amplification on four haplotype A strains. On gBlocks for LAMP-A, the lowest number of copies for a positive LAMP reaction was 60 on haplotype A and 600 on haplotype B; for LAMP-B, the lowest number of copies for a positive LAMP reaction was 60 on haplotype B and 600 on haplotype A. Therefore, considering the convenience of the LAMP technique, as well as the high specificity and sensitivity, the LAMP-A and LAMP-B primers can be used together to test the probable Lso-infected plant or psyllid samples to rapidly, accurately, and directly differentiate haplotypes A and B. We highly recommend this LAMP system to plant pathology practitioners and diagnostic labs for routine detection of Lso and confirmation of zebra chip disease on potato or tomato.